Gene-regulating compositions and methods for improved immunotherapy

ABSTRACT

The present disclosure provides methods and compositions related to the modification of immune effector cells to increase therapeutic efficacy. In some embodiments, immune effector cells modified to reduce expression of one or more endogenous target genes, or to reduce one or more functions of an endogenous protein to enhance effector functions of the immune cells are provided. In some embodiments, immune effector cells further modified by introduction of transgenes conferring antigen specificity, such as exogenous T cell receptors (TCRs) or chimeric antigen receptors (CARs) are provided. Methods of treating a cell proliferative disorder, such as a cancer, using the modified immune effector cells described herein are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/354,102, filed Mar. 14, 2019, which claims priority to U.S.Provisional Application Nos. 62/643,578, filed Mar. 15, 2018;62/643,587, filed Mar. 15, 2018; 62/643,597, filed Mar. 15, 2018;62/643,598, filed Mar. 15, 2018; 62/692,010, filed Jun. 29, 2018;62/692,019, filed Jun. 29, 2018; 62/692,100, filed Jun. 29, 2018;62/692,110, filed Jun. 29, 2018; 62/768,428, filed Nov. 16, 2018;62/768,443, filed Nov. 16, 2018; 62/768,448, filed Nov. 16, 2018;62/768,458, filed Nov. 16, 2018; and 62/804,265, filed Feb. 12, 2019,each of which is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:616582_KSQW-007CON_ST25.txt; date recorded: Feb. 14, 2020; file size 308kilobytes).

FIELD

The disclosure relates to methods, compositions, and components forediting a target nucleic acid sequence, or modulating expression of atarget nucleic acid sequence, and applications thereof in connectionwith immunotherapy, including use with receptor-engineered immuneeffector cells, in the treatment of cell proliferative diseases,inflammatory diseases, and/or infectious diseases.

BACKGROUND

Adoptive cell transfer utilizing genetically modified T cells, inparticular CAR-T cells has entered clinical testing as a therapeutic forsolid and hematologic malignancies. Results to date have been mixed. Inhematologic malignancies (especially lymphoma, CLL and ALL), themajority of patients in several Phase 1 and 2 trials exhibited at leasta partial response, with some exhibiting complete responses(Kochenderfer et al., 2012 Blood 1 19, 2709-2720). In 2017, the FDAapproved two CAR-T therapies, Kymriah™ and Yescarta™ both for thetreatment of hematological cancers. However, in most tumor types(including melanoma, renal cell carcinoma and colorectal cancer), fewerresponses have been observed (Johnson et al., 2009 Blood 1 14, 535-546;Lamers et al., 2013 Mol. Ther. 21, 904-912; Warren et al., 1998 CancerGene Ther. 5, S1-S2). As such, there is considerable room forimprovement with adoptive T cell therapies, as success has largely beenlimited to CAR-T cells approaches targeting hematological malignanciesof the B cell lineage.

SUMMARY

There exists a need to improve the efficacy of adoptive transfer ofmodified immune cells in cancer treatment, in particular increasing theefficacy of adoptive cell therapies against solid malignancies, asreduced responses have been observed in these tumor types (melanoma,renal cell carcinoma and colorectal cancer; Yong, 2017, Imm Cell Biol.,95:356-363). In addition, even in hematological malignancies where abenefit of adoptive transfer has been observed, not all patients respondand relapses occur with a greater than desired frequency, likely as aresult of diminished function of the adoptively transferred T cells.

Factors limiting the efficacy of genetically modified immune cells ascancer therapeutics include (1) cell proliferation, e.g., limitedproliferation of T cells following adoptive transfer; (2) cell survival,e.g., induction of T cell apoptosis by factors in the tumor environment;and (3) cell function, e.g., inhibition of cytotoxic T cell function byinhibitory factors secreted by host immune cells and cancer cells andexhaustion of immune cells during manufacturing processes and/or aftertransfer.

Particular features thought to increase the anti-tumor effects of animmune cell include a cell's ability to 1) proliferate in the hostfollowing adoptive transfer; 2) infiltrate a tumor; 3) persist in thehost and/or exhibit resistance to immune cell exhaustion; and 4)function in a manner capable of killing tumor cells. The presentdisclosure provides immune cells comprising decreased expression and/orfunction of one or more endogenous target genes wherein the modifiedimmune cells demonstrate an enhancement of one or more effectorfunctions including increased proliferation, increased infiltration intotumors, persistence of the immune cells in a subject, and/or increasedresistance to immune cell exhaustion. The present disclosure alsoprovides methods and compositions for modification of immune effectorcells to elicit enhanced immune cell activity towards a tumor cell, aswell as methods and compositions suitable for use in the context ofadoptive immune cell transfer therapy.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of ZC3H12Anucleic acid or protein (also known as Regnase-1). The presentdisclosure describes and demonstrates inhibition of ZC3H12A by multiplemodalities, including CRISPR/Cas systems, zinc-finger systems, andsiRNA/shRNA systems. In some embodiments, the reducedexpression/function of ZC3H12A is mediated by an antibody, a smallmolecule, or a peptide. In some embodiments, the present disclosureprovides a method of killing a cancerous cell comprising exposing thecancerous cell to a ZC3H12A protein inhibitor, wherein said inhibitor isan antibody, small molecule or peptide that binds to ZC3H12A and reducesZC3H12A function and wherein said inhibitor is in an amount effective tokill said cancerous cell. In some embodiments, the exposure is in vitro,in vivo, or ex vivo

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingexpression and/or function of one or more endogenous target genesselected from: (a) the group consisting of BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, andGNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3. In some embodiments, thereduced expression and/or function of the one or more endogenous genesenhances an effector function of the immune effector cell.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of one or more endogenous target genesselected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1,and BCOR. In some embodiments, the reduced expression and/or function ofthe one or more endogenous genes enhances an effector function of theimmune effector cell. In some embodiments, the gene-regulating system iscapable of reducing the expression and/or function of two or more ofendogenous target genes selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of theendogenous target genes is selected from the group consisting of IKZFIKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, and IKZF2 and at least one of the endogenous targetgenes is selected from the group consisting of CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.

In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of at least one endogenous target geneselected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS,RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS andat least one endogenous target gene selected from the group consistingof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.

In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of ZC3H12A and at least one endogenoustarget gene selected from the group consisting of IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, the gene-regulating system iscapable of reducing the expression and/or function of ZC3H12A and CBLB.In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of ZC3H12A and BCOR. In some embodiments,the gene-regulating system is capable of reducing the expression and/orfunction of ZC3H12A and TNFAIP3.

In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of MAP4K1 and at least one endogenoustarget gene selected from the group consisting of IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, the gene-regulating system iscapable of reducing the expression and/or function of MAP4K1 and CBLB.In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of MAP4K1 and BCOR. In some embodiments,the gene-regulating system is capable of reducing the expression and/orfunction of MAP4K1 and TNFAPI3.

In some embodiments, the gene-regulating system is capable of reducingthe expression and/or function of NR4A3 and at least one endogenoustarget gene selected from the group consisting of IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, the gene-regulating system iscapable of reducing the expression and/or function of NR4A3 and CBLB. Insome embodiments, the gene-regulating system is capable of reducing theexpression and/or function of NR4A3 and BCOR. In some embodiments, thegene-regulating system is capable of reducing the expression and/orfunction of NR4A3 and TNFAPI3.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system, wherein thegene-regulating system comprises (i) one or more nucleic acid molecules;(ii) one or more enzymatic proteins; or (iii) one or more guide nucleicacid molecules and an enzymatic protein. In some embodiments, the one ormore nucleic acid molecules are selected from an siRNA, an shRNA, amicroRNA (miR), an antagomiR, or an antisense RNA. In some embodiments,the gene-regulating system comprises an siRNA or an shRNA nucleic acidmolecule.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system, wherein thegene-regulating system comprises an siRNA or an shRNA nucleic acidmolecule, wherein the siRNA or shRNA molecule comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequencedefined by a set of genome coordinates shown in Table 5A and Table 5B.In some embodiments, the siRNA or shRNA comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 154-813. In someembodiments, the gene-regulating system comprises an siRNA or an shRNAnucleic acid molecule, wherein the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6A andTable 6B. In some embodiments, the siRNA or shRNA comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 814-1064.

In some embodiments, the gene-regulating system comprises an siRNA or anshRNA nucleic acid molecule, wherein the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table6C and Table 6D. In some embodiments, the siRNA or shRNA comprises about19-30 nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 1065-1509. In someembodiments, the gene-regulating system comprises an siRNA or an shRNAnucleic acid molecule, wherein the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6E andTable 6F. In some embodiments, the siRNA or shRNA comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 510-1538. In someembodiments, the gene-regulating system comprises an siRNA or an shRNAnucleic acid molecule, wherein the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6G andTable 6H. In some embodiments, the siRNA or shRNA comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 1539-1566.

In some embodiments, the gene-regulating system comprises a plurality ofsiRNA or shRNA molecules and is capable of reducing the expressionand/or function of two or more endogenous target genes. In someembodiments, at least one of the endogenous target genes is selectedfrom the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39,SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and at leastone of the endogenous target genes is selected from the group consistingof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, atleast one of the plurality of siRNA or shRNA molecules comprises about19-30 nucleotides that bind to an RNA sequence encoded by a DNA sequencedefined by a set of genome coordinates shown in Table 6A and Table 6Band at least one of the plurality of siRNA or shRNA molecules comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 5A andTable 5B. In some embodiments, at least one of the plurality of siRNA orshRNA molecules comprises about 19-30 nucleotides that bind to an RNAsequence encoded by a DNA sequence selected from the group consisting ofSEQ ID NOs: 814-1064 and at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-813. In some embodiments, at least one of the endogenous targetgenes is selected from the group consisting of BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,and GNAS and at least one of the endogenous target genes is selectedfrom the group consisting of TNFAIP3, CBLB, and BCOR.

In some embodiments, the gene-regulating system comprises a plurality ofsiRNA or shRNA molecules and is capable of reducing the expressionand/or function of two or more endogenous target genes. In someembodiments, at least one of the endogenous target genes is ZC3H12A andat least one of the endogenous target genes is selected from the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments,at least one of the plurality of siRNA or shRNA molecules comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6C andTable 6D and at least one of the plurality of siRNA or shRNA moleculescomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table5A and Table 5B. In some embodiments, at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 1065-1509 and at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 154-813. In some embodiments, at least one ofthe endogenous target genes is selected from the group consisting ofZC3H12A and at least one of the endogenous target genes is selected fromthe group consisting of TNFAIP3, CBLB, and BCOR.

In some embodiments, the gene-regulating system comprises a plurality ofsiRNA or shRNA molecules and is capable of reducing the expressionand/or function of two or more endogenous target genes. In someembodiments, at least one of the endogenous target genes is MAP4K1 andat least one of the endogenous target genes is selected from the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments,at least one of the plurality of siRNA or shRNA molecules comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6E andTable 6F and at least one of the plurality of siRNA or shRNA moleculescomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table5A and Table 5B. In some embodiments, at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 510-1538 and at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 154-813. In some embodiments, at least one ofthe endogenous target genes is MAP4K1 and at least one of the endogenoustarget genes is selected from the group consisting of TNFAIP3, CBLB, andBCOR.

In some embodiments, the gene-regulating system comprises a plurality ofsiRNA or shRNA molecules and is capable of reducing the expressionand/or function of two or more endogenous target genes. In someembodiments, at least one of the endogenous target genes is NR4A3 and atleast one of the endogenous target genes is selected from the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments,at least one of the plurality of siRNA or shRNA molecules comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6G andTable 6H and at least one of the plurality of siRNA or shRNA moleculescomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table5A and Table 5B. In some embodiments, at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 1539-1566 and at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 154-813. In some embodiments, at least one ofthe endogenous target genes is NR4A3 and at least one of the endogenoustarget genes is selected from the group consisting of TNFAIP3, CBLB, andBCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system, wherein thegene-regulating system comprises an enzymatic protein, and wherein theenzymatic protein has been engineered to specifically bind to a targetsequence in one or more of the endogenous genes. In some embodiments,the protein is a Transcription activator-like effector nuclease (TALEN),a zinc-finger nuclease, or a meganuclease.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system, wherein thegene-regulating system comprises a guide nucleic acid molecule and anenzymatic protein, wherein the nucleic acid molecule is a guide RNA(gRNA) molecule and the enzymatic protein is a Cas protein or Casortholog.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least one endogenous target gene,wherein the gene-regulating system comprises a guide RNA (gRNA) moleculeand a Cas protein or Cas ortholog, and wherein the one or moreendogenous target genes is selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR, and wherein the gRNA molecule comprises atargeting domain sequence that binds to a nucleic acid sequence definedby a set of genome coordinates shown in Table 5A and Table 5B. In someembodiments, the gRNA molecule comprises a targeting domain sequencethat binds to a target DNA sequence selected from the group consistingof SEQ ID NOs: 154-813. In some embodiments, the gRNA molecule comprisesa targeting domain sequence encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 154-813.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least one endogenous target gene,wherein the gene-regulating system comprises a guide RNA (gRNA) moleculeand a Cas protein or Cas ortholog, and wherein the one or moreendogenous target genes is selected from the group consisting ofBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS, and wherein the gRNA molecule comprisesa targeting domain sequence that binds to a nucleic acid sequencedefined by a set of genome coordinates shown in Table 6A and Table 6B.In some embodiments, the gRNA molecule comprises a targeting domainsequence that binds to a target DNA sequence selected from the groupconsisting of SEQ ID NOs: 814-1064. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 814-1064.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least one endogenous target gene,wherein the gene-regulating system comprises a guide RNA (gRNA) moleculeand a Cas protein or Cas ortholog, and wherein the one or moreendogenous target genes is ZC3H12A, and wherein the gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Table 6C andTable 6D. In some embodiments, the gRNA molecule comprises a targetingdomain sequence that binds to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1065-1509. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1065-1509.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least one endogenous target gene,wherein the gene-regulating system comprises a guide RNA (gRNA) moleculeand a Cas protein or Cas ortholog, and wherein the one or moreendogenous target genes is MAP4K1, and wherein the gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Tables 6E and6F. In some embodiments, the gRNA molecule comprises a targeting domainsequence that binds to a target DNA sequence selected from the groupconsisting of SEQ ID NOs: 510-1538. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 510-1538.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least one endogenous target gene,wherein the gene-regulating system comprises a guide RNA (gRNA) moleculeand a Cas protein or Cas ortholog, and wherein the one or moreendogenous target genes is NR4A3, and wherein the gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Tables 6G and6H. In some embodiments, the gRNA molecule comprises a targeting domainsequence that binds to a target DNA sequence selected from the groupconsisting of SEQ ID NOs: 1539-1566. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1539-1566.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least two endogenous target genes,wherein the gene-regulating system comprises a plurality of gRNAs and aCas protein or Cas ortholog. In some embodiments, the present disclosureprovides a modified immune effector cell comprising a gene-regulatingsystem capable of reducing the expression and/or function of at leasttwo endogenous target genes, wherein the gene-regulating systemcomprises a plurality of gRNAs and a Cas protein or Cas ortholog,wherein at least one of the endogenous target genes selected from thegroup consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A,CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and at least one ofthe endogenous target genes is selected from the group consisting ofIKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a nucleic acid sequence defined by a setof genome coordinates shown in Table 6A and Table 6B and at least one ofthe plurality of gRNA molecule comprises a targeting domain sequencethat binds to a nucleic acid sequence defined by a set of genomecoordinates shown in Table 5A and Table 5B. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 814-1064 and at least one of theplurality of gRNA molecules comprises a targeting domain sequence thatbinds to a target DNA sequence selected from the group consisting of SEQID NOs: 154-813. In some embodiments, at least one of the plurality ofgRNA molecules comprises a targeting domain sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NOs:814-1064 and at least one of the plurality of gRNA molecules comprises atargeting domain sequence encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 154-813. In some embodiments,at least one of the endogenous target genes selected from the groupconsisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and at least one of theendogenous target genes is selected from the group consisting ofTNFAIP3, CBLB, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least two endogenous target genes,wherein the gene-regulating system comprises a plurality of gRNAs and aCas protein or Cas ortholog, wherein at least one of the endogenoustarget genes is ZC3H12A and at least one of the endogenous target genesis selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, at least one of the plurality ofgRNA molecules comprises a targeting domain sequence that binds to anucleic acid sequence defined by a set of genome coordinates shown inTable 6C and Table 6D and at least one of the plurality of gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Table 5A andTable 5B. In some embodiments, at least one of the plurality of gRNAmolecules comprises a targeting domain sequence that binds to a targetDNA sequence selected from the group consisting of SEQ ID NOs: 1065-1509and at least one of the plurality of gRNA molecules comprises atargeting domain sequence that binds to a target DNA sequence selectedfrom the group consisting of SEQ ID NOs: 154-813. In some embodiments,at least one of the plurality of gRNA molecules comprises a targetingdomain sequence encoded by a DNA sequence selected from the groupconsisting of SEQ ID NOs: 1065-1509 and at least one of the plurality ofgRNA molecules comprises a targeting domain sequence encoded by a DNAsequence selected from the group consisting of SEQ ID NOs: 154-813. Insome embodiments, at least one of the endogenous target genes is ZC3H12Aand at least one of the endogenous target genes is selected from thegroup consisting of TNFAIP3, CBLB, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least two endogenous target genes,wherein the gene-regulating system comprises a plurality of gRNAs and aCas protein or Cas ortholog, wherein at least one of the endogenoustarget genes is MAP4K1 and at least one of the endogenous target genesis selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, at least one of the plurality ofgRNA molecules comprises a targeting domain sequence that binds to anucleic acid sequence defined by a set of genome coordinates shown inTable 6E and Table 6F and at least one of the plurality of gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Table 5A andTable 5B. In some embodiments, at least one of the plurality of gRNAmolecules comprises a targeting domain sequence that binds to a targetDNA sequence selected from the group consisting of SEQ ID NOs: 510-1538and at least one of the plurality of gRNA molecules comprises atargeting domain sequence that binds to a target DNA sequence selectedfrom the group consisting of SEQ ID NOs: 154-813. In some embodiments,at least one of the plurality of gRNA molecules comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 510-1538 and at least one of theplurality of gRNA molecules comprises a targeting domain sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-813. In some embodiments, at least one of the endogenous targetgenes is MAP4K1 and at least one of the endogenous target genes isselected from the group consisting of TNFAIP3, CBLB, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a gene-regulating system capable of reducingthe expression and/or function of at least two endogenous target genes,wherein the gene-regulating system comprises a plurality of gRNAs and aCas protein or Cas ortholog, wherein at least one of the endogenoustarget genes is NR4A3 and at least one of the endogenous target genes isselected from the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1,and BCOR. In some embodiments, at least one of the plurality of gRNAmolecules comprises a targeting domain sequence that binds to a nucleicacid sequence defined by a set of genome coordinates shown in Table 6Gand Table 6H and at least one of the plurality of gRNA moleculecomprises a targeting domain sequence that binds to a nucleic acidsequence defined by a set of genome coordinates shown in Table 5A andTable 5B. In some embodiments, at least one of the plurality of gRNAmolecules comprises a targeting domain sequence that binds to a targetDNA sequence selected from the group consisting of SEQ ID NOs: 1539-1566and at least one of the plurality of gRNA molecules comprises atargeting domain sequence that binds to a target DNA sequence selectedfrom the group consisting of SEQ ID NOs: 154-813. In some embodiments,at least one of the plurality of gRNA molecules comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1539-1566 and at least one of theplurality of gRNA molecules comprises a targeting domain sequenceencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 154-813. In some embodiments, at least one of the endogenoustarget genes is NR4A3 and at least one of the endogenous target genes isselected from the group consisting of TNFAIP3, CBLB, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising a Cas protein or Cas ortholog, wherein: (a) theCas protein is a wild-type Cas protein comprising two enzymaticallyactive domains, and capable of inducing double stranded DNA breaks; (b)the Cas protein is a Cas nickase mutant comprising one enzymaticallyactive domain and capable of inducing single stranded DNA breaks; or (c)the Cas protein is a deactivated Cas protein (dCas) and is associatedwith a heterologous protein capable of modulating the expression of theone or more endogenous target genes. In some embodiments, the Casprotein is a Cas9 protein. In some embodiments, the heterologous proteinis selected from the group consisting of MAX-interacting protein 1(MXI1), Krüppel-associated box (KRAB) domain, methyl-CpG binding protein2 (MECP2), and four concatenated mSin3 domains (SID4X).

In some embodiments, the gene regulating system introduces aninactivating mutation into the one or more endogenous target genes. Insome embodiments, the inactivating mutation comprises a deletion,substitution, or insertion of one or more nucleotides in the genomicsequences of the two or more endogenous genes. In some embodiments, thedeletion is a partial or complete deletion of the two or more endogenoustarget genes. In some embodiments, the inactivating mutation is a frameshift mutation. In some embodiments, the inactivating mutation reducesthe expression and/or function of the two or more endogenous targetgenes.

In some embodiments, the gene-regulating system is introduced to theimmune effector cell by transfection, transduction, electroporation, orphysical disruption of the cell membrane by a microfluidics device. Insome embodiments, the gene-regulating system is introduced as apolynucleotide encoding one or more components of the system, a protein,or a ribonucleoprotein (RNP) complex.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of one ormore endogenous genes selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR, wherein the reduced expression and/orfunction of the one or more endogenous genes enhances an effectorfunction of the immune effector cell. In some embodiments, the presentdisclosure provides a modified immune effector cell comprising reducedexpression and/or function of one or more endogenous genes selectedfrom: (a) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, andIKZF2; or (b) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3,TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein the reduced expressionand/or function of the one or more endogenous genes enhances an effectorfunction of the immune effector cell.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of one ormore endogenous genes selected from: (a) the group consisting ofBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3,wherein the reduced expression and/or function of the one or moreendogenous genes enhances an effector function of the modified immuneeffector cell.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of two ormore target genes selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1,and BCOR, wherein the reduced expression and/or function of the two ormore endogenous genes enhances an effector function of the modifiedimmune effector cell. In some embodiments, the modified immune effectorcomprises reduced expression and/or function of CBLB and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of two ormore target genes, wherein at least one target gene is selected from thegroup consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A,CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and wherein atleast one target gene is selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR, wherein the reduced expression and/orfunction of the two or more endogenous genes enhances an effectorfunction of the modified immune effector cell.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of two ormore target genes, wherein at least one target gene is ZC3H12A, andwherein at least one target gene is selected from the group consistingof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein the reducedexpression and/or function of the two or more endogenous genes enhancesan effector function of the modified immune effector cell. In someembodiments, the modified immune effector comprises reduced expressionand/or function of ZC3H12A and CBLB. In some embodiments, the modifiedimmune effector comprises reduced expression and/or function of ZC3H12Aand BCOR. In some embodiments, the modified immune effector comprisesreduced expression and/or function of ZC3H12A and TNFAIP3.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of two ormore target genes, wherein at least one target gene is MAP4K1, andwherein at least one target gene is selected from the group consistingof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein the reducedexpression and/or function of the two or more endogenous genes enhancesan effector function of the modified immune effector cell. In someembodiments, the modified immune effector comprises reduced expressionand/or function of MAP4K1 and CBLB. In some embodiments, the modifiedimmune effector comprises reduced expression and/or function of MAP4K1and BCOR. In some embodiments, the modified immune effector comprisesreduced expression and/or function of MAP4K1 and TNFAIP3.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising reduced expression and/or function of two ormore target genes, wherein at least one target gene is NR4A3, andwherein at least one target gene is selected from the group consistingof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein the reducedexpression and/or function of the two or more endogenous genes enhancesan effector function of the modified immune effector cell. In someembodiments, the modified immune effector comprises reduced expressionand/or function of NR4A3 and CBLB. In some embodiments, the modifiedimmune effector comprises reduced expression and/or function of NR4A3and BCOR. In some embodiments, the modified immune effector comprisesreduced expression and/or function of NR4A3 and TNFAIP3.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in one or moreendogenous genes selected from the group consisting of IKZF1, IKZF3,GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3,RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4,PTPN6, PDCD1, and BCOR. In some embodiments, the present disclosureprovides a modified immune effector cell comprising an inactivatingmutation in one or more endogenous genes selected from: (a) the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2; or (b) the groupconsisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in one or moreendogenous genes selected from: (a) the group consisting of BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in two or more targetgenes selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA,SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D,NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In someembodiments, the modified immune effector comprises an inactivatingmutation in the CBLB and BCOR genes.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in two or more targetgenes, wherein at least one target gene is selected from the groupconsisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS, and at least one targetgene is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in two or more targetgenes, wherein at least one target gene is ZC3H12A and at least onetarget gene is selected from the group consisting of IKZF1, IKZF3,GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3,RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4,PTPN6, PDCD1, and BCOR. In some embodiments, the modified immuneeffector comprises an inactivating mutation in the ZC3H12A and CBLBgenes. In some embodiments, the modified immune effector comprises aninactivating mutation in the ZC3H12A and BCOR genes. In someembodiments, the modified immune effector comprises an inactivatingmutation in the ZC3H12A and TNFAIP3 genes.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in two or more targetgenes, wherein at least one target gene is MAP4K1 and at least onetarget gene is selected from the group consisting of IKZF1, IKZF3,GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3,RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4,PTPN6, PDCD1, and BCOR. In some embodiments, the modified immuneeffector comprises an inactivating mutation in the MAP4K1 and CBLBgenes. In some embodiments, the modified immune effector comprises aninactivating mutation in the MAP4K1 and BCOR genes. In some embodiments,the modified immune effector comprises an inactivating mutation in theMAP4K1 and TNFAIP3 genes.

In some embodiments, the present disclosure provides a modified immuneeffector cell comprising an inactivating mutation in two or more targetgenes, wherein at least one target gene is NR4A3 and at least one targetgene is selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, the modified immune effectorcomprises an inactivating mutation in the NR4A3 and CBLB genes. In someembodiments, the modified immune effector comprises an inactivatingmutation in the NR4A3 and BCOR genes. In some embodiments, the modifiedimmune effector comprises an inactivating mutation in the NR4A3 andTNFAIP3 genes.

In some embodiments, the inactivating mutation comprises a deletion,substitution, or insertion of one or more nucleotides in the genomicsequences of the two or more endogenous genes. In some embodiments, thedeletion is a partial or complete deletion of the two or more endogenoustarget genes. In some embodiments, the inactivating mutation is a frameshift mutation. In some embodiments, the inactivating mutation reducesthe expression and/or function of the two or more endogenous targetgenes.

In some embodiments, the expression of the one or more endogenous targetgenes is reduced by at least 50%, at least 60%, at least 70%, at least80%, or at least 90% compared to an un-modified or control immuneeffector cell. In some embodiments, the function of the one or moreendogenous target genes is reduced by at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% compared to an un-modified orcontrol immune effector cell.

In some embodiments, the modified immune effector cell further comprisesan engineered immune receptor displayed on the cell surface. In someembodiments, the engineered immune receptor is a CAR comprising anantigen-binding domain, a transmembrane domain, and an intracellularsignaling domain. In some embodiments, the engineered immune receptor isan engineered TCR. In some embodiments, the engineered immune receptorspecifically binds to an antigen expressed on a target cell, wherein theantigen is a tumor-associated antigen.

In some embodiments, the modified immune effector cell further comprisesan exogenous transgene expressing an immune activating molecule. In someembodiments, the immune activating molecule is selected from the groupconsisting of a cytokine, a chemokine, a co-stimulatory molecule, anactivating peptide, an antibody, or an antigen-binding fragment thereof.In some embodiments, the antibody or binding fragment thereofspecifically binds to and inhibits the function of the protein encodedby NRP1, HAVCR2, LAG3, TIGIT, CTLA4, or PDCD1.

In some embodiments, the immune effector cell is a wherein the immuneeffector cell is a lymphocyte selected from a T cell, a natural killer(NK) cell, an NKT cell. In some embodiments, the lymphocyte is a tumorinfiltrating lymphocyte (TIL).

In some embodiments, the effector function is selected from cellproliferation, cell viability, tumor infiltration, cytotoxicity,anti-tumor immune responses, and/or resistance to exhaustion.

In some embodiments, the present disclosure provides a compositioncomprising the modified immune effector cells described herein. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier or diluent. In some embodiments, the compositioncomprises at least 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, or1×10¹¹ modified immune effector cells. In some embodiments, thecomposition is suitable for administration to a subject in need thereof.In some embodiments, the composition comprises autologous immuneeffector cells derived from the subject in need thereof. In someembodiments, the composition comprises allogeneic immune effector cellsderived from a donor subject.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression and/or function of one or moreendogenous target genes in a cell selected from: (a) the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2; or (b) the groupconsisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR, wherein the system comprises (i) a nucleic acidmolecule; (ii) an enzymatic; or (iii) a guide nucleic acid molecule andan enzymatic protein.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell selected from: (a) the group consisting of BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3, whereinthe system comprises (i) a nucleic acid molecule; (ii) an enzymatic; or(iii) a guide nucleic acid molecule and an enzymatic protein.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target genes areselected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2 or is selected fromCBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCORand wherein the gRNA molecule comprises a targeting domain sequence thatbinds to a target DNA sequence defined by a set of genomic coordinatesshown in Table 5A and Table 5B. In some embodiments, the one or moreendogenous target genes are selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, and IKZF2 and wherein the gRNA molecule comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 154-498. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 154-498.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target genes areselected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR and wherein the gRNA molecule comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 499-813. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target genes areselected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and wherein the gRNAmolecule comprises a targeting domain sequence that binds to a targetDNA sequence defined by a set of genomic coordinates shown in Table 6Aand Table 6B. In some embodiments, the gRNA molecule comprises atargeting domain sequence that binds to a target DNA sequence selectedfrom the group consisting of SEQ ID NOs: 814-1064. In some embodiments,the gRNA molecule comprises a targeting domain sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NOs:814-1064.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target gene isZC3H12A and wherein the gRNA molecule comprises a targeting domainsequence that binds to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6C and Table 6D. In some embodiments, thegRNA molecule comprises a targeting domain sequence that binds to atarget DNA sequence selected from the group consisting of SEQ ID NOs:1065-1509. In some embodiments, the gRNA molecule comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1065-1509. In some embodiments, the gRNAmolecule comprises a targeting domain sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1065-1509.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target genescomprises MAP4K1 and wherein the gRNA molecule comprises a targetingdomain sequence that binds to a target DNA sequence defined by a set ofgenomic coordinates shown in Table 6E and Table 6F. In some embodiments,the gRNA molecule comprises a targeting domain sequence that binds to atarget DNA sequence selected from the group consisting of SEQ ID NOs:510-1538. In some embodiments, the gRNA molecule comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 510-1538.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell comprising a guide RNA (gRNA) nucleic acid molecule anda Cas endonuclease, wherein the one or more endogenous target genescomprises NR4A3 and wherein the gRNA molecule comprises a targetingdomain sequence that binds to a target DNA sequence defined by a set ofgenomic coordinates shown in Table 6G and Table 6H. In some embodiments,the gRNA molecule comprises a targeting domain sequence that binds to atarget DNA sequence selected from the group consisting of SEQ ID NOs:1539-1566. In some embodiments, the gRNA molecule comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1539-1566.

In some embodiments, the gene-regulating system comprises an siRNA or anshRNA nucleic acid molecule.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell, wherein the gene-regulating system comprises an siRNAor an shRNA nucleic acid molecule and wherein the one or more endogenoustarget genes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, andIKZF2 or is selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR and wherein the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table5A and Table 5B. In some embodiments, the one or more endogenous targetgenes are selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2 andwherein the siRNA or shRNA molecule comprises about 19-30 nucleotidesthat bind to an RNA sequence encoded by a DNA sequence selected from SEQID NOs: 154-498. In some embodiments, the one or more endogenous targetgenes are selected from CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4,PTPN6, PDCD1, and BCOR and wherein the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence selected from SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell, wherein the gene-regulating system comprises an siRNAor an shRNA nucleic acid molecule, wherein the one or more endogenoustarget genes are selected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39,SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and whereinthe siRNA or shRNA molecule comprises about 19-30 nucleotides that bindto an RNA sequence encoded by a DNA sequence defined by a set of genomecoordinates shown in Table 6A and Table 6B. In some embodiments, thesiRNA or shRNA molecule comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence selected from SEQ ID NOs:814-1064.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell, wherein the gene-regulating system comprises an siRNAor an shRNA nucleic acid molecule, wherein the one or more endogenoustarget gene is ZC3H12A and wherein the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence defined by a set of genome coordinates shown in Table 6C andTable 6D. In some embodiments, the siRNA or shRNA molecule comprisesabout 19-30 nucleotides that bind to an RNA sequence encoded by a DNAsequence selected from SEQ ID NOs: 1065-1509.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell, wherein the gene-regulating system comprises an siRNAor an shRNA nucleic acid molecule, wherein the one or more endogenoustarget genes comprises MAP4K1 and wherein the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table6E and Table 6F. In some embodiments, the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence selected from SEQ ID NOs: 510-1538.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing expression of one or more endogenous targetgenes in a cell, wherein the gene-regulating system comprises an siRNAor an shRNA nucleic acid molecule, wherein the one or more endogenoustarget genes comprises NR4A3 and wherein the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence defined by a set of genome coordinates shown in Table6G and Table 6H. In some embodiments, the siRNA or shRNA moleculecomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence selected from SEQ ID NOs: 1539-1566.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein at least one of theendogenous target genes is selected from: (a) the group consisting ofBCL2L11, FLI1, CALM2, DHODH, (IMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c) MAP4K1; or (d) NR4A3,and wherein at least one of the endogenous target genes is selectedfrom: (e) the group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, andIKZF2; or (f) the group consisting of CBLB, PPP2R2D, NRP1, HAVCR2, LAG3,TIGIT, CTLA4, PTPN6, PDCD1, and BCOR, wherein the system comprises (i) anucleic acid molecule; (ii) an enzymatic; or (iii) a guide nucleic acidmolecule and an enzymatic protein

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the system comprises aplurality of guide RNA (gRNA) nucleic acid molecules and a Casendonuclease.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the system comprises aplurality of guide RNA (gRNA) nucleic acid molecules and a Casendonuclease, wherein at least one of the endogenous target genes isselected from the group consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS,RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS andat least one of the endogenous target genes is selected from the groupconsisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments,at least one of the plurality of gRNAs binds to a target DNA sequencedefined by a set of genomic coordinates shown in Table 6A and Table 6B,and wherein at least one of the plurality of gRNAs binds to a target DNAsequence defined by a set of genomic coordinates shown in Table 5A andTable 5B. In some embodiments, at least one of the plurality of gRNAmolecules comprises a targeting domain sequence that binds to a targetDNA sequence selected from the group consisting of SEQ ID NOs: 814-1064and wherein at least one of the plurality of gRNA molecules comprises atargeting domain sequence that binds to a target DNA sequence selectedfrom the group consisting of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.In some embodiments, at least one of the plurality of gRNA moleculescomprises a targeting domain sequence encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 814-1064 and whereinat least one of the plurality of gRNA molecules comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from SEQ IDNOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the system comprises aplurality of guide RNA (gRNA) nucleic acid molecules and a Casendonuclease, wherein at least one of the endogenous target genes isZC3H12A and at least one of the endogenous target genes is selected fromthe group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. Insome embodiments, at least one of the plurality of gRNAs binds to atarget DNA sequence defined by a set of genomic coordinates shown inTable 6C and Table 6D, and wherein at least one of the plurality ofgRNAs binds to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A and Table 5B. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 1065-1509 and wherein at least one ofthe plurality of gRNA molecules comprises a targeting domain sequencethat binds to a target DNA sequence selected from the group consistingof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 1065-1509 and wherein at least one ofthe plurality of gRNA molecules comprises a targeting domain sequencethat binds to a target DNA sequence selected from the group consistingof SEQ ID NOs: 499-524. In some embodiments, at least one of theplurality of gRNA molecules comprises a targeting domain sequenceencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1065-1509 and wherein at least one of the plurality of gRNAmolecules comprises a targeting domain sequence encoded by a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 154-498or SEQ ID NOs: 499-813. In some embodiments, at least one of theplurality of gRNA molecules comprises a targeting domain sequenceencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1065-1509 and wherein at least one of the plurality of gRNAmolecules comprises a targeting domain sequence encoded by a nucleicacid sequence selected from SEQ ID NOs: 499-524.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the system comprises aplurality of guide RNA (gRNA) nucleic acid molecules and a Casendonuclease, wherein at least one of the endogenous target genes isMAP4K1 and at least one of the endogenous target genes is selected fromthe group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAGS, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. Insome embodiments, at least one of the plurality of gRNAs binds to atarget DNA sequence defined by a set of genomic coordinates shown inTable 6E and Table 6F, and wherein at least one of the plurality ofgRNAs binds to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A and Table 5B. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 510-1538 and wherein at least one of theplurality of gRNA molecules comprises a targeting domain sequence thatbinds to a target DNA sequence selected from the group consisting of SEQID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at leastone of the plurality of gRNA molecules comprises a targeting domainsequence that binds to a target DNA sequence selected from the groupconsisting of SEQ ID NOs: 510-1538 and wherein at least one of theplurality of gRNA molecules comprises a targeting domain sequence thatbinds to a target DNA sequence selected from the group consisting of SEQID NOs: 499-524. In some embodiments, at least one of the plurality ofgRNA molecules comprises a targeting domain sequence encoded by anucleic acid sequence selected from the group consisting of SEQ ID NOs:510-1538 and wherein at least one of the plurality of gRNA moleculescomprises a targeting domain sequence encoded by a nucleic acid sequenceselected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In someembodiments, at least one of the plurality of gRNA molecules comprises atargeting domain sequence encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 510-1538 and wherein at leastone of the plurality of gRNA molecules comprises a targeting domainsequence encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 499-524.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the system comprises aplurality of guide RNA (gRNA) nucleic acid molecules and a Casendonuclease, wherein at least one of the endogenous target genes isNR4A3 and at least one of the endogenous target genes is selected fromthe group consisting of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR. Insome embodiments, at least one of the plurality of gRNAs binds to atarget DNA sequence defined by a set of genomic coordinates shown inTable 6G and Table 6H, and wherein at least one of the plurality ofgRNAs binds to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A and Table 5B. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 1539-1566 and wherein at least one ofthe plurality of gRNA molecules comprises a targeting domain sequencethat binds to a target DNA sequence selected from the group consistingof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, atleast one of the plurality of gRNA molecules comprises a targetingdomain sequence that binds to a target DNA sequence selected from thegroup consisting of SEQ ID NOs: 1539-1566 and wherein at least one ofthe plurality of gRNA molecules comprises a targeting domain sequencethat binds to a target DNA sequence selected from the group consistingof SEQ ID NOs: 499-524. In some embodiments, at least one of theplurality of gRNA molecules comprises a targeting domain sequenceencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1539-1566 and wherein at least one of the plurality of gRNAmolecules comprises a targeting domain sequence encoded by a nucleicacid sequence selected from SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.In some embodiments, at least one of the plurality of gRNA moleculescomprises a targeting domain sequence encoded by a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1539-1566 and whereinat least one of the plurality of gRNA molecules comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises a Cas protein,wherein the Cas protein is: (a) a wild-type Cas protein comprising twoenzymatically active domains, and capable of inducing double strandedDNA breaks; (b) a Cas nickase mutant comprising one enzymatically activedomain and capable of inducing single stranded DNA breaks; (c) adeactivated Cas protein (dCas) and is associated with a heterologousprotein capable of modulating the expression of the one or moreendogenous target genes. In some embodiments, the heterologous proteinis selected from the group consisting of MAX-interacting protein 1(MXI1), Krüppel-associated box (KRAB) domain, and four concatenatedmSin3 domains (SID4X). In some embodiments, the Cas protein is a Cas9protein.

In some embodiments, the gene-regulating system comprises a nucleic acidmolecule and wherein the nucleic acid molecule is an siRNA, an shRNA, amicroRNA (miR), an antagomiR, or an antisense RNA. In some embodiments,the gene-regulating system comprises a plurality of shRNA or siRNAmolecules.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the gene-regulating systemcomprises a plurality of shRNA or siRNA molecules, wherein at least oneof the endogenous target genes is selected from the group consisting ofBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS and at least one of the endogenous targetgenes is selected from the group consisting of IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, and BCOR. In some embodiments, at least one of the plurality ofsiRNA or shRNA molecules comprises about 19-30 nucleotides that bind toan RNA sequence encoded by a DNA sequence defined by a set of genomecoordinates shown in Table 6A and Table 6B and at least one of theplurality of siRNA or shRNA molecules comprises about 19-30 nucleotidesthat bind to an RNA sequence encoded by a DNA sequence defined by a setof genome coordinates shown in Table 5A and Table 5B. In someembodiments, at least one of the plurality of siRNA or shRNA moleculescomprises about 19-30 nucleotides that bind to an RNA sequence encodedby a DNA sequence selected from the group consisting of SEQ ID NOs:814-1064 and wherein at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the gene-regulating systemcomprises a plurality of shRNA or siRNA molecules, wherein at least oneof the endogenous target genes is ZC3H12A and at least one of theendogenous target genes is selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of theplurality of siRNA or shRNA molecules comprises about 19-30 nucleotidesthat bind to an RNA sequence encoded by a DNA sequence defined by a setof genome coordinates shown in Table 6C and Table 6D and at least one ofthe plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequencedefined by a set of genome coordinates shown in Table 5A and Table 5B.In some embodiments, at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 1065-1509 and at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least oneof the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 1065-1509 and at leastone of the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 499-524.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the gene-regulating systemcomprises a plurality of shRNA or siRNA molecules, wherein at least oneof the endogenous target genes is MAP4K1 and at least one of theendogenous target genes is selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of theplurality of siRNA or shRNA molecules comprises about 19-30 nucleotidesthat bind to an RNA sequence encoded by a DNA sequence defined by a setof genome coordinates shown in Table 6E and Table 6F and at least one ofthe plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequencedefined by a set of genome coordinates shown in Table 5A and Table 5B.In some embodiments, at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 510-1538 and at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least oneof the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 510-1538 and at leastone of the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 499-524.

In some embodiments, the present disclosure provides a gene-regulatingsystem capable of reducing the expression and/or function of two or moreendogenous target genes in a cell, wherein the gene-regulating systemcomprises a plurality of shRNA or siRNA molecules, wherein at least oneof the endogenous target genes is NR4A3 and at least one of theendogenous target genes is selected from the group consisting of IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR. In some embodiments, at least one of theplurality of siRNA or shRNA molecules comprises about 19-30 nucleotidesthat bind to an RNA sequence encoded by a DNA sequence defined by a setof genome coordinates shown in Table 6G and Table 6H and at least one ofthe plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequencedefined by a set of genome coordinates shown in Table 5A and Table 5B.In some embodiments, at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 1539-1566 and at least one of the plurality of siRNA or shRNAmolecules comprises about 19-30 nucleotides that bind to an RNA sequenceencoded by a DNA sequence selected from the group consisting of SEQ IDNOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, at least oneof the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 1539-1566 and at leastone of the plurality of siRNA or shRNA molecules comprises about 19-30nucleotides that bind to an RNA sequence encoded by a DNA sequenceselected from the group consisting of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises a proteincomprising a DNA binding domain and an enzymatic domain and is selectedfrom a zinc finger nuclease and a transcription-activator-like effectornuclease (TALEN).

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising a vector encoding one or more gRNAs and a vectorencoding a Cas endonuclease protein, wherein the one or more gRNAscomprise a targeting domain sequence encoded by a nucleic acid sequenceselected from: SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs:510-1538, SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ ID NOs:499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising a vector encoding a plurality of gRNAs and a vectorencoding a Cas endonuclease protein, wherein at least one of theplurality of gRNA comprises a targeting domain sequence encoded by anucleic acid sequence selected from: SEQ ID NOs: 814-1064, SEQ ID NOs:1065-1509, SEQ ID NOs: 510-1538, or SEQ ID NOs: 1539-1566, and whereinat least one of the plurality of gRNA comprises a targeting domainsequence encoded by a nucleic acid sequence selected from: SEQ ID NOs:154-498 or SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising a vector encoding one or more gRNAs and an mRNAmolecule encoding a Cas endonuclease protein, wherein the one or moregRNAs comprise a targeting domain sequence encoded by a nucleic acidsequence selected from SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ IDNOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising a vector encoding a plurality of gRNAs and an mRNAmolecule encoding a Cas endonuclease protein, wherein at least one ofthe plurality of gRNA comprises a targeting domain sequence encoded by anucleic acid sequence selected from: SEQ ID NOs: 814-1064, SEQ ID NOs:1065-1509, SEQ ID NOs: 510-1538, or SEQ ID NOs: 1539-1566, and whereinat least one of the plurality of gRNA comprises a targeting domainsequence encoded by a nucleic acid sequence selected from: SEQ ID NOs:154-498 or SEQ ID NOs: 499-813.

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising one or more gRNAs and a Cas endonuclease protein,wherein the one or more gRNAs comprise a targeting domain sequenceencoded by a nucleic acid sequence selected from: SEQ ID NOs: 814-1064,SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQID NOs: 154-498, or SEQ ID NOs: 499-813, and wherein the one or moregRNAs and the Cas endonuclease protein are complexed to form aribonucleoprotein (RNP) complex.

In some embodiments, the present disclosure provides a gene-regulatingsystem comprising a plurality of gRNAs and a Cas endonuclease protein:wherein at least one of the plurality of gRNA comprises a targetingdomain sequence encoded by a nucleic acid sequence selected from: SEQ IDNOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538, or SEQ IDNOs: 1539-1566, wherein at least one of the plurality of gRNA comprisesa targeting domain sequence encoded by a nucleic acid sequence selectedfrom: SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813, and wherein the one ormore gRNAs and the Cas endonuclease protein are complexed to form aribonucleoprotein (RNP) complex.

In some embodiments, the present disclosure provides a kit comprising agene-regulating system described herein.

In some embodiments, the present disclosure provides a gRNA nucleic acidmolecule comprising a targeting domain nucleic acid sequence that bindsto a target sequence in an endogenous target gene selected from: (a) thegroup consisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A,CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS; (b) ZC3H12A; (c)MAP4K1; (d) NR4A3; (e) IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA,SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, and IKZF2; or (f)CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR.In some embodiments, (a) the endogenous gene is selected from the groupconsisting of BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and the gRNA comprises atargeting domain sequence that binds to a target DNA sequence located atgenomic coordinates selected from those shown in Tables 6A and 6B; (b)the endogenous gene is ZC3H12A and the gRNA comprises a targeting domainsequence that binds to a target DNA sequence located at genomiccoordinates selected from those shown in Table 6C and Table 6D; (c) theendogenous gene is MAP4K1 and the gRNA comprises a targeting domainsequence that binds to a target DNA sequence located at genomiccoordinates selected from those shown in Table 6E and Table 6F; (d) theendogenous gene is NR4A3 and the gRNA comprises a targeting domainsequence that binds to a target DNA sequence located at genomiccoordinates selected from those shown in Table 6G and Table 6H; (e) theendogenous gene is selected from the group consisting of IKZF1, IKZF3,GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3,RC3H1, TRAF6, and IKZF2 and the gRNA comprises a targeting domainsequence that binds to a target DNA sequence located at genomiccoordinates selected from those shown in Table 5A and Table 5B; or (f)the endogenous gene is selected from the group consisting of CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR andthe gRNA comprises a targeting domain sequence that binds to a targetDNA sequence located at genomic coordinates selected from those shown inTable 5A and Table 5B.

In some embodiments, the gRNA comprises a targeting domain sequence thatbinds to a target DNA sequence selected from SEQ ID NOs: 814-1064, SEQID NOs: 1065-1509, SEQ ID NOs: 510-1538, SEQ ID NOs: 1539-1566, SEQ IDNOs: 154-498, or SEQ ID NOs: 499-813. In some embodiments, the gRNAcomprises a targeting domain sequence encoded by a sequence selectedfrom SEQ ID NOs: 814-1064, SEQ ID NOs: 1065-1509, SEQ ID NOs: 510-1538,SEQ ID NOs: 1539-1566, SEQ ID NOs: 154-498, or SEQ ID NOs: 499-813. Insome embodiments, the target sequence comprises a PAM sequence.

In some embodiments, the gRNA is a modular gRNA molecule. In someembodiments, the gRNA is a dual gRNA molecule. In some embodiments, thetargeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or morenucleotides in length. In some embodiments, the gRNA molecule comprisesa modification at or near its 5′ end (e.g., within 1-10, 1-5, or 1-2nucleotides of its 5′ end) and/or a modification at or near its 3′ end(e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3′ end). In someembodiments, the modified gRNA exhibits increased stability towardsnucleases when introduced into a T cell. In some embodiments, themodified gRNA exhibits a reduced innate immune response when introducedinto a T cell.

In some embodiments, the present disclosure provides a polynucleotidemolecule encoding a gRNA molecule described herein. In some embodiments,the present disclosure provides a composition comprising one or moregRNA molecules described herein or polynucleotides encoding the same. Insome embodiments, the present disclosure provides a kit comprising oneor more gRNA molecules described herein or polynucleotides encoding thesame.

In some embodiments, the present disclosure provides a method ofproducing a modified immune effector cell comprising: (a) obtaining animmune effector cell from a subject; (b) introducing the gene-regulatingsystem into the immune effector cell; and (c) culturing the immuneeffector cell such that the expression and/or function of one or moreendogenous target genes is reduced compared to an immune effector cellthat has not been modified.

In some embodiments, the present disclosure provides a method ofproducing a modified immune effector cell comprising introducing agene-regulating system described herein into the immune effector cell.In some embodiments, the methods further comprise introducing apolynucleotide sequence encoding an engineered immune receptor selectedfrom a CAR and a TCR. In some embodiments, the gene-regulating systemand/or the polynucleotide encoding the engineered immune receptor areintroduced to the immune effector cell by transfection, transduction,electroporation, or physical disruption of the cell membrane by amicrofluidics device. In some embodiments, the gene-regulating system isintroduced as a polynucleotide sequence encoding one or more componentsof the system, as a protein, or as an ribonucleoprotein (RNP) complex.

In some embodiments, the present disclosure provides a method ofproducing a modified immune effector cell comprising: (a) expanding apopulation of immune effector cells in culture; and (b) introducing agene-regulating system into the population of immune effector cells. Insome embodiments, the methods further comprise obtaining the populationof immune effector cells from a subject. In some embodiments, thegene-regulating system is introduced to the population of immuneeffector cells before, during, or after expansion. In some embodiments,the expansion of the population of immune effector cells comprises afirst round expansion and a second round of expansion. In someembodiments, the gene-regulating system is introduced to the populationof immune effector cells before, during, or after the first round ofexpansion. In some embodiments, the gene-regulating system is introducedto the population of immune effector cells before, during, or after thesecond round of expansion. In some embodiments, the gene-regulatingsystem is introduced to the population of immune effector cells beforethe first and second rounds of expansion. In some embodiments, thegene-regulating system is introduced to the population of immuneeffector cells after the first and second rounds of expansion.

In some embodiments, the present disclosure provides a method oftreating a disease or disorder in a subject in need thereof comprisingadministering an effective amount of a modified immune effectordescribed herein, or composition thereof. In some embodiments, thedisease or disorder is a cell proliferative disorder, an inflammatorydisorder, or an infectious disease. In some embodiments, the disease ordisorder is a cancer or a viral infection. In some embodiments, thecancer is selected from a leukemia, a lymphoma, or a solid tumor. Insome embodiments, the solid tumor is a melanoma, a pancreatic tumor, abladder tumor, a lung tumor or metastasis, a colorectal cancer, or ahead and neck cancer. In some embodiments, the cancer is a PD1 resistantor insensitive cancer. In some embodiments, the subject has previouslybeen treated with a PD1 inhibitor or a PDL1 inhibitor. In someembodiments, the methods further comprise administering to the subjectan antibody or binding fragment thereof that specifically binds to andinhibits the function of the protein encoded by NRP1, HAVCR2, LAGS,TIGIT, CTLA4, or PDCD1. In some embodiments, the modified immuneeffector cells are autologous to the subject. In some embodiments, themodified immune effector cells are allogenic to the subject. In someembodiments, the subject has not undergone lymphodepletion prior toadministration of the modified immune effector cells or compositionsthereof. In some embodiments, the subject does not receive high-doseIL-2 treatment with or after the administration of the modified immuneeffector cells or compositions thereof. In some embodiments, the subjectreceives low-dose IL-2 treatment with or after the administration of themodified immune effector cells or compositions thereof. In someembodiments, the subject does not receive IL-2 treatment with or afterthe administration of the modified immune effector cells or compositionsthereof.

In some embodiments, the present disclosure provides a method of killinga cancerous cell comprising exposing the cancerous cell to a modifiedimmune effector cell described herein or a composition thereof. In someembodiments, the exposure is in vitro, in vivo, or ex vivo.

In some embodiments, the present disclosure provides a method ofenhancing one or more effector functions of an immune effector cellcomprising introducing a gene-regulating system described herein intothe immune effector cell. In some embodiments, the present disclosureprovides a method of enhancing one or more effector functions of animmune effector cell comprising introducing a gene-regulating systemdescribed herein into the immune effector cell, wherein the modifiedimmune effector cell demonstrates one or more enhanced effectorfunctions compared to the immune effector cell that has not beenmodified. In some embodiments, the one or more effector functions areselected from cell proliferation, cell viability, cytotoxicity, tumorinfiltration, increased cytokine production, anti-tumor immuneresponses, and/or resistance to exhaustion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-FIG. 1B illustrate combinations of endogenous target genes thatcan be modified by the methods described herein.

FIG. 2A-FIG. 2B illustrate combinations of endogenous target genes thatcan be modified by the methods described herein.

FIG. 3A-FIG. 3B illustrate combinations of endogenous target genes thatcan be modified by the methods described herein.

FIG. 4A-FIG. 4D illustrates editing of the TRAC and B2M genes usingmethods described herein.

FIG. 5A-FIG. 5B illustrate TIDE analysis data for editing of CBLB inprimary human T cells.

FIG. 6 illustrates a western blot for CBLB protein in primary human Tcells edited with a CBLB gRNA (D6551-CBLB) compared to unedited controls(D6551-WT).

FIG. 7A-FIG. 7E show tumor growth over time in a murine B16/Ovasyngeneic tumor model. FIG. 7A shows tumor growth in mice treated withCBLB-edited OT1 T cells compared to unedited OT1 T cells. FIG. 7B-FIG.7C shows tumor growth in mice treated with ZC3H12A-edited OT1 T cellscompared to unedited OT1 T cells. FIG. 7D shows tumor growth in micetreated with MAP4K1-edited OT1 T cells compared to unedited OT1 T cells.FIG. 7E shows tumor growth in mice treated with NR4A3-edited OT1 T cellscompared to unedited OT1 T cells.

FIG. 8A-FIG. 8B show tumor growth over time in a murine B16/Ovasyngeneic tumor model in mice treated with single-edited T cells and/orin combination with anti-PD1 therapy. FIG. 8A shows tumor growth in micetreated with a combination of MAP4K1-edited OT1 T cells and anti-PD1therapy compared to control and MAP4K1-edited OT1 cells alone.

FIG. 8B shows tumor growth in mice treated with a combination ofNR4A3-edited OT1 T cells and anti-PD1 therapy compared to control andNR4A3-edited OT1 cells alone.

FIG. 9 shows tumor growth over time in a murine MC38/gp100 syngeneictumor model in mice treated with Zc3h12a-edited PMEL T cells.

FIG. 10 shows a survival curve of mice treated with Zc3h12a-edited PMELT cells and PD1-edited PMEL T cells in a B16F10 lung met model.

FIG. 11 shows tumor growth over time in a murine Eg7 Ova syngeneic tumormodel in mice treated with Zc3h12a-edited T cells.

FIG. 12A-FIG. 12B shows tumor growth over time in a murine A375xenograft model for mice. FIG. 12A shows tumor growth over time in amurine A375 xenograft model for mice treated with CBLB-editedNY-ESO-1-specific TCR transgenic T cells compared to uneditedNY-ESO-1-specific TCR transgenic T cells. FIG. 12B shows tumor growthover time in a murine A375 xenograft model for mice treated withZC3H12A-edited NY-ESO-1-specific TCR transgenic T cells compared tocontrol edited T cells.

FIG. 13A-FIG. 13B shows tumor growth after treatment with single-editedCD19 CART cells in a subcutaneous model of Burkitt's lymphoma using Rajicells. FIG. 13A shows tumor growth after treatment with MAP4K1-editedCD19 CAR T cells compared to unedited CD19 CART cells. FIG. 13B showstumor growth after treatment with NR4A3-edited CD19 CART cells comparedto unedited CD19 CART cells.

FIG. 14 shows tumor growth over time in mice treated with BCOR-edited,CBLB-edited, or BCOR/CBLB dual-edited anti-CD19 CART cells. Tumor growthis compared to mice treated with no CAR T cells or unedited anti-CD19CAR T cells.

FIG. 15 shows accumulation of BCOR-edited or BCOR/CBLB-edited CD19 CAR Tcells in an in vitro culture system.

FIG. 16 shows IL-2 production by BCOR-edited or BCOR/CBLB-edited CD19CAR T cells in an in vitro culture system.

FIG. 17 shows IFNγ production by BCOR-edited or BCOR/CBLB-edited CD19CAR T cells in an in vitro culture system.

FIG. 18 shows tumor growth over time in a murine B16/Ova syngeneic tumormodel in mice treated with dual-edited Zc3h12a/Cblb OT1 T cells.

FIG. 19 shows tumor growth over time in a murine B16/Ova syngeneic tumormodel in mice treated with Pd1/Lag3 dual-edited OT1 T cells.

FIG. 20 shows validation of Zc3h12a as target conferring anti-tumormemory and epitope spreading.

FIG. 21 shows production of IFNγ, IL-2, and TNFα in MAP4K1-edited PBMCs.

FIG. 22 shows mRNA expression of Icos, Il6, Il2, Ifng, and Nfkbiz inZc3h12a-edited murine CD8 T cells.

FIG. 23 shows cell surface expression of ICOS in Zc3h12a-edited murineCD8 T cells.

FIG. 24 shows production of IL-2 and IFNγ by Zc3h12a-edited murine CD8 Tcells after anti-CD3/CD28 stimulation.

FIG. 25A-FIG. 25B show increased expression of IL6 in ZC3H12A-editedprimary human T cells. FIG. 25A shows IL-6 protein production fromZC3H12A-edited PBMCs derived from donors. FIG. 25B shows IL6 mRNAexpression in ZC3H12A-edited PBMCs.

DETAILED DESCRIPTION

The present disclosure provides methods and compositions related to themodification of immune effector cells to increase their therapeuticefficacy in the context of immunotherapy. In some embodiments, immuneeffector cells are modified by the methods of the present disclosure toreduce expression of one or more endogenous target genes, or to reduceone or more functions of an endogenous protein such that one or moreeffector functions of the immune cells are enhanced. In someembodiments, the immune effector cells are further modified byintroduction of transgenes conferring antigen specificity, such asintroduction of T cell receptor (TCR) or chimeric antigen receptor (CAR)expression constructs. In some embodiments, the present disclosureprovides compositions and methods for modifying immune effector cells,such as compositions of gene-regulating systems. In some embodiments,the present disclosure provides methods of treating a cell proliferativedisorder, such as a cancer, comprising administration of the modifiedimmune effector cells described herein to a subject in need thereof.

I. Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

As used in this specification, the term “and/or” is used in thisdisclosure to mean either “and” or “or” unless indicated otherwise.

Throughout this specification, unless the context requires otherwise,the words “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Decrease” or “reduce” refers to a decrease or a reduction in aparticular value of at least 5%, for example, a 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, or 100% decrease as compared to a reference value. Adecrease or reduction in a particular value may also be represented as afold-change in the value compared to a reference value, for example, atleast a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold,or more, decrease as compared to a reference value.

“Increase” refers to an increase in a particular value of at least 5%,for example, a 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 200%,300%, 400%, 500%, or more increase as compared to a reference value. Anincrease in a particular value may also be represented as a fold-changein the value compared to a reference value, for example, at least a 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more,increase as compared to the level of a reference value.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. “Oligonucleotide”generally refers to polynucleotides of between about 5 and about 100nucleotides of single- or double-stranded DNA. However, for the purposesof this disclosure, there is no upper limit to the length of anoligonucleotide. Oligonucleotides are also known as “oligomers” or“oligos” and may be isolated from genes, or chemically synthesized bymethods known in the art. The terms “polynucleotide” and “nucleic acid”should be understood to include, as applicable to the embodiments beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

“Fragment” refers to a portion of a polypeptide or polynucleotidemolecule containing less than the entire polypeptide or polynucleotidesequence. In some embodiments, a fragment of a polypeptide orpolynucleotide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, or 99% of the entire length of thereference polypeptide or polynucleotide. In some embodiments, apolypeptide or polynucleotide fragment may contain 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, or more nucleotides or amino acids.

The term “sequence identity” refers to the percentage of bases or aminoacids between two polynucleotide or polypeptide sequences that are thesame, and in the same relative position. As such one polynucleotide orpolypeptide sequence has a certain percentage of sequence identitycompared to another polynucleotide or polypeptide sequence. For sequencecomparison, typically one sequence acts as a reference sequence, towhich test sequences are compared. The term “reference sequence” refersto a molecule to which a test sequence is compared.

“Complementary” refers to the capacity for pairing, through basestacking and specific hydrogen bonding, between two sequences comprisingnaturally or non-naturally occurring bases or analogs thereof. Forexample, if a base at one position of a nucleic acid is capable ofhydrogen bonding with a base at the corresponding position of a target,then the bases are considered to be complementary to each other at thatposition. Nucleic acids can comprise universal bases, or inert abasicspacers that provide no positive or negative contribution to hydrogenbonding. Base pairings may include both canonical Watson-Crick basepairing and non-Watson-Crick base pairing (e.g., Wobble base pairing andHoogsteen base pairing). It is understood that for complementary basepairings, adenosine-type bases (A) are complementary to thymidine-typebases (T) or uracil-type bases (U), that cytosine-type bases (C) arecomplementary to guanosine-type bases (G), and that universal bases suchas such as 3-nitropyrrole or 5-nitroindole can hybridize to and areconsidered complementary to any A, C, U, or T. Nichols et al., Nature,1994; 369:492-493 and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also been considered in the art to be auniversal base and is considered complementary to any A, C, U, or T. SeeWatkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

As referred to herein, a “complementary nucleic acid sequence” is anucleic acid sequence comprising a sequence of nucleotides that enablesit to non-covalently bind to another nucleic acid in asequence-specific, antiparallel, manner (i.e., a nucleic acidspecifically binds to a complementary nucleic acid) under theappropriate in vitro and/or in vivo conditions of temperature andsolution ionic strength.

Methods of sequence alignment for comparison and determination ofpercent sequence identity and percent complementarity are well known inthe art. Optimal alignment of sequences for comparison can be conducted,e.g., by the homology alignment algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology), by use of algorithms know in the art including theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,(1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol.Biol. 215:403-410, respectively. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation.

Herein, the term “hybridize” refers to pairing between complementarynucleotide bases (e.g., adenine (A) forms a base pair with thymine (T)in a DNA molecule and with uracil (U) in an RNA molecule, and guanine(G) forms a base pair with cytosine (C) in both DNA and RNA molecules)to form a double-stranded nucleic acid molecule. (See, e.g., Wahl andBerger (1987) Methods Enzymol. 152:399; Kimmel, (1987) Methods Enzymol.152:507). In addition, it is also known in the art that forhybridization between two RNA molecules (e.g., dsRNA), guanine (G) basepairs with uracil (U). For example, G/U base-pairing is partiallyresponsible for the degeneracy (i.e., redundancy) of the genetic code inthe context of tRNA anti-codon base-pairing with codons in mRNA. In thecontext of this disclosure, a guanine (G) of a protein-binding segment(dsRNA duplex) of a guide RNA molecule is considered complementary to auracil (U), and vice versa. As such, when a G/U base-pair can be made ata given nucleotide position a protein-binding segment (dsRNA duplex) ofa guide RNA molecule, the position is not considered to benon-complementary, but is instead considered to be complementary. It isunderstood in the art that the sequence of polynucleotide need not be100% complementary to that of its target nucleic acid to be specificallyhybridizable. Moreover, a polynucleotide may hybridize over one or moresegments such that intervening or adjacent segments are not involved inthe hybridization event (e.g., a loop structure or hairpin structure). Apolynucleotide can comprise at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, or 100% sequence complementarity to a targetregion within the target nucleic acid sequence to which they aretargeted.

The term “modified” refers to a substance or compound (e.g., a cell, apolynucleotide sequence, and/or a polypeptide sequence) that has beenaltered or changed as compared to the corresponding unmodified substanceor compound.

The term “naturally-occurring” as used herein as applied to a nucleicacid, a polypeptide, a cell, or an organism, refers to a nucleic acid,polypeptide, cell, or organism that is found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by a human in the laboratoryis naturally occurring.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.

An “expression cassette” or “expression construct” refers to a DNApolynucleotide sequence operably linked to a promoter. “Operably linked”refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Forinstance, a promoter is operably linked to a polynucleotide sequence ifthe promoter affects the transcription or expression of thepolynucleotide sequence.

The term “recombinant vector” as used herein refers to a polynucleotidemolecule capable transferring or transporting another polynucleotideinserted into the vector. The inserted polynucleotide may be anexpression cassette. In some embodiments, a recombinant vector may beviral vector or a non-viral vector (e.g., a plasmid).

The term “sample” refers to a biological composition (e.g., a cell or aportion of a tissue) that is subjected to analysis and/or geneticmodification. In some embodiments, a sample is a “primary sample” inthat it is obtained directly from a subject; in some embodiments, a“sample” is the result of processing of a primary sample, for example toremove certain components and/or to isolate or purify certain componentsof interest.

The term “subject” includes animals, such as e.g. mammals. In someembodiments, the mammal is a primate. In some embodiments, the mammal isa human. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; or domesticated animals such asdogs and cats. In some embodiments (e.g., particularly in researchcontexts) subjects are rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like. The terms “subject”and “patient” are used interchangeably herein.

“Administration” refers herein to introducing an agent or compositioninto a subject.

“Treating” as used herein refers to delivering an agent or compositionto a subject to affect a physiologic outcome.

As used herein, the term “effective amount” refers to the minimum amountof an agent or composition required to result in a particularphysiological effect. The effective amount of a particular agent may berepresented in a variety of ways based on the nature of the agent, suchas mass/volume, # of cells/volume, particles/volume, (mass of theagent)/(mass of the subject), # of cells/(mass of subject), orparticles/(mass of subject). The effective amount of a particular agentmay also be expressed as the half-maximal effective concentration(EC₅₀), which refers to the concentration of an agent that results in amagnitude of a particular physiological response that is half-waybetween a reference level and a maximum response level.

“Population” of cells refers to any number of cells greater than 1, butis preferably at least 1×10³ cells, at least 1×10⁴ cells, at least 1×10⁵cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least 1×10⁸ cells,at least 1×10⁹ cells, at least 1×10¹⁰ cells, at least 1×10¹¹ or morecells. A population of cells may refer to an in vitro population (e.g.,a population of cells in culture) or an in vivo population (e.g., apopulation of cells residing in a particular tissue).

General methods in molecular and cellular biochemistry can be found insuch standard textbooks as Molecular Cloning: A Laboratory Manual, 3rdEd. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols inMolecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); NonviralVectors for Gene Therapy (Wagner et al. eds., Academic Press 1999);Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); ImmunologyMethods Manual (I. Lefkovits ed., Academic Press 1997); and Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, John Wiley & Sons 1998), the disclosures of which areincorporated herein by reference.

II. Modified Immune Effector Cells

In some embodiments, the present disclosure provides modified immuneeffector cells. Herein, the term “modified immune effector cells”encompasses immune effector cells comprising one or more genomicmodifications resulting in the reduced expression and/or function of oneor more endogenous target genes as well as immune effector cellscomprising a gene-regulating system capable of reducing the expressionand/or function of one or more endogenous target genes. Herein, an“un-modified immune effector cell” or “control immune effector cell”refers to a cell or population of cells wherein the genomes have notbeen modified and that does not comprise a gene-regulating system orcomprises a control gene-regulating system (e.g., an empty vectorcontrol, a non-targeting gRNA, a scrambled siRNA, etc.).

The term “immune effector cell” refers to cells involved in mountinginnate and adaptive immune responses, including but not limited tolymphocytes (such as T-cells (including thymocytes) and B-cells),natural killer (NK) cells, NKT cells, macrophages, monocytes,eosinophils, basophils, neutrophils, dendritic cells, and mast cells. Insome embodiments, the modified immune effector cell is a T cell, such asa CD4+ T cell, a CD8+ T cell (also referred to as a cytotoxic T cell orCTL), a regulatory T cell (Treg), a Th1 cell, a Th2 cell, or a Th17cell.

In some embodiments, the immune effector cell is a T cell that has beenisolated from a tumor sample (referred to herein as “tumor infiltratinglymphocytes” or “TILs”). Without wishing to be bound by theory, it isthought that TILs possess increased specificity to tumor antigens(Radvanyi et al., 2012 Clin Canc Res 18:6758-6770) and can thereforemediate tumor antigen-specific immune response (e.g., activation,proliferation, and cytotoxic activity against the cancer cell) leadingto cancer cell destruction (Brudno et al., 2018 Nat Rev Clin Onc15:31-46)) without the introduction of an exogenous engineered receptor.Therefore, in some embodiments, TILs are isolated from a tumor in asubject, expanded ex vivo, and re-infused into a subject. In someembodiments, TILs are modified to express one or more exogenousreceptors specific for an autologous tumor antigen, expanded ex vivo,and re-infused into the subject. Such embodiments can be modeled usingin vivo mouse models wherein mice have been transplanted with a cancercell line expressing a cancer antigen (e.g., CD19) and are treated withmodified T cells that express an exogenous receptor that is specific forthe cancer antigen (See e.g., Examples 10 and 11).

In some embodiments, the immune effector cell is an animal cell or isderived from an animal cell, including invertebrate animals andvertebrate animals (e.g., fish, amphibian, reptile, bird, or mammal). Insome embodiments, the immune effector cell is a mammalian cell or isderived from a mammalian cell (e.g., a pig, a cow, a goat, a sheep, arodent, a non-human primate, a human, etc.). In some embodiments, theimmune effector cell is a rodent cell or is derived from a rodent cell(e.g., a rat or a mouse). In some embodiments, the modified immuneeffector cell is a human cell or is derived from a human cell.

In some embodiments, the modified immune effector cells comprise one ormore modifications (e.g., insertions, deletions, or mutations of one ormore nucleic acids) in the genomic DNA sequence of an endogenous targetgene resulting in the reduced expression and/or function the endogenousgene. Such modifications are referred to herein as “inactivatingmutations” and endogenous genes comprising an inactivating mutation arereferred to as “modified endogenous target genes.” In some embodiments,the inactivating mutations reduce or inhibit mRNA transcription, therebyreducing the expression level of the encoded mRNA transcript andprotein. In some embodiments, the inactivating mutations reduce orinhibit mRNA translation, thereby reducing the expression level of theencoded protein. In some embodiments, the inactivating mutations encodea modified endogenous protein with reduced or altered function comparedto the unmodified (i.e., wild-type) version of the endogenous protein(e.g., a dominant-negative mutant, described infra).

In some embodiments, the modified immune effector cells comprise one ormore genomic modifications at a genomic location other than anendogenous target gene that result in the reduced expression and/orfunction of the endogenous target gene or that result in the expressionof a modified version of an endogenous protein. For example, in someembodiments, a polynucleotide sequence encoding a gene regulating systemis inserted into one or more locations in the genome, thereby reducingthe expression and/or function of an endogenous target gene upon theexpression of the gene-regulating system. In some embodiments, apolynucleotide sequence encoding a modified version of an endogenousprotein is inserted at one or more locations in the genome, wherein thefunction of the modified version of the protein is reduced compared tothe un-modified or wild-type version of the protein (e.g., adominant-negative mutant, described infra).

In some embodiments, the modified immune effector cells described hereincomprise one or more modified endogenous target genes, wherein the oneor more modifications result in a reduced expression and/or function ofa gene product (i.e., an mRNA transcript or a protein) encoded by theendogenous target gene compared to an unmodified immune effector cell.For example, in some embodiments, a modified immune effector celldemonstrates reduced expression of an mRNA transcript and/or reducedexpression of a protein. In some embodiments, the expression of the geneproduct in a modified immune effector cell is reduced by at least 5%compared to the expression of the gene product in an unmodified immuneeffector cell. In some embodiments, the expression of the gene productin a modified immune effector cell is reduced by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more compared to the expression of thegene product in an unmodified immune effector cell. In some embodiments,the modified immune effector cells described herein demonstrate reducedexpression and/or function of gene products encoded by a plurality(e.g., two or more) of endogenous target genes compared to theexpression of the gene products in an unmodified immune effector cell.For example, in some embodiments, a modified immune effector celldemonstrates reduced expression and/or function of gene products from 2,3, 4, 5, 6, 7, 8, 9, 10, or more endogenous target genes compared to theexpression of the gene products in an unmodified immune effector cell.

In some embodiments, the present disclosure provides a modified immuneeffector cell wherein one or more endogenous target genes, or a portionthereof, are deleted (i.e., “knocked-out”) such that the modified immuneeffector cell does not express the mRNA transcript or protein. In someembodiments, a modified immune effector cell comprises deletion of aplurality of endogenous target genes, or portions thereof. In someembodiments, a modified immune effector cell comprises deletion of 2, 3,4, 5, 6, 7, 8, 9, 10, or more endogenous target genes.

In some embodiments, the modified immune effector cells described hereincomprise one or more modified endogenous target genes, wherein the oneor more modifications to the target DNA sequence result in expression ofa protein with reduced or altered function (e.g., a “modified endogenousprotein”) compared to the function of the corresponding proteinexpressed in an unmodified immune effector cell (e.g., a “unmodifiedendogenous protein”). In some embodiments, the modified immune effectorcells described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or moremodified endogenous target genes encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore modified endogenous proteins. In some embodiments, the modifiedendogenous protein demonstrates reduced or altered binding affinity foranother protein expressed by the modified immune effector cell orexpressed by another cell; reduced or altered signaling capacity;reduced or altered enzymatic activity; reduced or altered DNA-bindingactivity; or reduced or altered ability to function as a scaffoldingprotein.

In some embodiments, the modified endogenous target gene comprises oneor more dominant negative mutations. As used herein, a“dominant-negative mutation” refers to a substitution, deletion, orinsertion of one or more nucleotides of a target gene such that theencoded protein acts antagonistically to the protein encoded by theunmodified target gene. The mutation is dominant-negative because thenegative phenotype confers genic dominance over the positive phenotypeof the corresponding unmodified gene. A gene comprising one or moredominant-negative mutations and the protein encoded thereby are referredto as a “dominant-negative mutants”, e.g. dominant-negative genes anddominant-negative proteins. In some embodiments, the dominant negativemutant protein is encoded by an exogenous transgene inserted at one ormore locations in the genome of the immune effector cell.

Various mechanisms for dominant negativity are known. Typically, thegene product of a dominant negative mutant retains some functions of theunmodified gene product but lacks one or more crucial other functions ofthe unmodified gene product. This causes the dominant-negative mutant toantagonize the unmodified gene product. For example, as an illustrativeembodiment, a dominant-negative mutant of a transcription factor maylack a functional activation domain but retain a functional DNA bindingdomain. In this example, the dominant-negative transcription factorcannot activate transcription of the DNA as the unmodified transcriptionfactor does, but the dominant-negative transcription factor canindirectly inhibit gene expression by preventing the unmodifiedtranscription factor from binding to the transcription-factor bindingsite. As another illustrative embodiment, dominant-negative mutations ofproteins that function as dimers are known. Dominant-negative mutants ofsuch dimeric proteins may retain the ability to dimerize with unmodifiedprotein but be unable to function otherwise. The dominant-negativemonomers, by dimerizing with unmodified monomers to form heterodimers,prevent formation of functional homodimers of the unmodified monomers.

In some embodiments, the modified immune effector cells comprise agene-regulating system capable of reducing the expression or function ofone or more endogenous target genes. The gene-regulating system canreduce the expression and/or function of the endogenous target genesmodifications by a variety of mechanisms including by modifying thegenomic DNA sequence of the endogenous target gene (e.g., by insertion,deletion, or mutation of one or more nucleic acids in the genomic DNAsequence); by regulating transcription of the endogenous target gene(e.g., inhibition or repression of mRNA transcription); and/or byregulating translation of the endogenous target gene (e.g., by mRNAdegradation).

In some embodiments, the modified immune effector cells described hereincomprise a gene-regulating system (e.g., a nucleic acid-basedgene-regulating system, a protein-based gene-regulating system, or acombination protein/nucleic acid-based gene-regulating system). In suchembodiments, the gene-regulating system comprised in the modified immuneeffector cell is capable of modifying one or more endogenous targetgenes. In some embodiments, the modified immune effector cells describedherein comprise a gene-regulating system comprising:

(a) one or more nucleic acid molecules capable of reducing theexpression or modifying the function of a gene product encoded by one ormore endogenous target genes;

(b) one or more polynucleotides encoding a nucleic acid molecule that iscapable of reducing the expression or modifying the function of a geneproduct encoded by one or more endogenous target genes;

(c) one or more proteins capable of reducing the expression or modifyingthe function of a gene product encoded by one or more endogenous targetgenes;

(d) one or more polynucleotides encoding a protein that is capable ofreducing the expression or modifying the function of a gene productencoded by one or more endogenous target genes;

(e) one or more guide RNAs (gRNAs) capable of binding to a target DNAsequence in an endogenous gene;

(f) one or more polynucleotides encoding one or more gRNAs capable ofbinding to a target DNA sequence in an endogenous gene;

(g) one or more site-directed modifying polypeptides capable ofinteracting with a gRNA and modifying a target DNA sequence in anendogenous gene;

(h) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gRNA and modifying a targetDNA sequence in an endogenous gene;

(i) one or more guide DNAs (gDNAs) capable of binding to a target DNAsequence in an endogenous gene;

(j) one or more polynucleotides encoding one or more gDNAs capable ofbinding to a target DNA sequence in an endogenous gene;

(k) one or more site-directed modifying polypeptides capable ofinteracting with a gDNA and modifying a target DNA sequence in anendogenous gene;

(l) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gDNA and modifying a targetDNA sequence in an endogenous gene;

(m) one or more gRNAs capable of binding to a target mRNA sequenceencoded by an endogenous gene;

(n) one or more polynucleotides encoding one or more gRNAs capable ofbinding to a target mRNA sequence encoded by an endogenous gene;

(o) one or more site-directed modifying polypeptides capable ofinteracting with a gRNA and modifying a target mRNA sequence encoded byan endogenous gene;

(p) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gRNA and modifying a targetmRNA sequence encoded by an endogenous gene; or

(q) any combination of the above.

In some embodiments, one or more polynucleotides encoding thegene-regulating system are inserted into the genome of the immuneeffector cell. In some embodiments, one or more polynucleotides encodingthe gene-regulating system are expressed episomaly and are not insertedinto the genome of the immune effector cell.

In some embodiments, the modified immune effector cells described hereincomprise reduced expression and/or function of one or more endogenoustarget genes and further comprise one or more exogenous transgenesinserted at one or more genomic loci (e.g., a genetic “knock-in”). Insome embodiments, the one or more exogenous transgenes encode detectabletags, safety-switch systems, chimeric switch receptors, and/orengineered antigen-specific receptors.

In some embodiments, the modified immune effector cells described hereinfurther comprise an exogenous transgene encoding a detectable tag.Examples of detectable tags include but are not limited to, FLAG tags,poly-histidine tags (e.g. 6×His), SNAP tags, Halo tags, cMyc tags,glutathione-S-transferase tags, avidin, enzymes, fluorescent proteins,luminescent proteins, chemiluminescent proteins, bioluminescentproteins, and phosphorescent proteins. In some embodiments thefluorescent protein is selected from the group consisting of blue/UVproteins (such as BFP, TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal,Sirius, Sapphire, and T-Sapphire); cyan proteins (such as CFP, eCFP,Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan,TagCFP, and mTFP1); green proteins (such as: GFP, eGFP, meGFP (A208Kmutation), Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2,mUKG, mWasabi, Clover, and mNeonGreen); yellow proteins (such as YFP,eYFP, Citrine, Venus, SYFP2, and TagYFP); orange proteins (such asMonomeric Kusabira-Orange, mKOκ, mKO2, mOrange, and mOrange2); redproteins (such as RFP, mRaspberry, mCherry, mStrawberry, mTangerine,tdTomato, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2); far-red proteins(such as mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP);near-infrared proteins (such as TagRFP657, IFP1.4, and iRFP); longstokes shift proteins (such as mKeima Red, LSS-mKate1, LSS-mKate2, andmBeRFP); photoactivatible proteins (such as PA-GFP, PAmCherryl, andPATagRFP); photoconvertible proteins (such as Kaede (green), Kaede(red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green),mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, and PSmOrange);and photoswitchable proteins (such as Dronpa). In some embodiments, thedetectable tag can be selected from AmCyan, AsRed, DsRed2, DsRedExpress, E2-Crimson, HcRed, ZsGreen, ZsYellow, mCherry, mStrawberry,mOrange, mBanana, mPlum, mRasberry, tdTomato, DsRed Monomer, and/orAcGFP, all of which are available from Clontech.

In some embodiments, the modified immune effector cells described hereinfurther comprise an exogenous transgene encoding a safety-switch system.Safety-switch systems (also referred to in the art as suicide genesystems) comprise exogenous transgenes encoding for one or more proteinsthat enable the elimination of a modified immune effector cell after thecell has been administered to a subject. Examples of safety-switchsystems are known in the art. For example, safety-switch systems includegenes encoding for proteins that convert non-toxic pro-drugs into toxiccompounds such as the Herpes simplex thymidine kinase (Hsv-tk) andganciclovir (GCV) system (Hsv-tk/GCV). Hsv-tk converts non-toxic GCVinto a cytotoxic compound that leads to cellular apoptosis. As such,administration of GCV to a subject that has been treated with modifiedimmune effector cells comprising a transgene encoding the Hsv-tk proteincan selectively eliminate the modified immune effector cells whilesparing endogenous immune effector cells. (See e.g., Bonini et al.,Science, 1997, 276(5319):1719-1724; Ciceri et al., Blood, 2007,109(11):1828-1836; Bondanza et al., Blood 2006, 107(5):1828-1836).

Additional safety-switch systems include genes encoding for cell-surfacemarkers, enabling elimination of modified immune effector cells byadministration of a monoclonal antibody specific for the cell-surfacemarker via ADCC. In some embodiments, the cell-surface marker is CD20and the modified immune effector cells can be eliminated byadministration of an anti-CD20 monoclonal antibody such as Rituximab(See e.g., Introna et al., Hum Gene Ther, 2000, 11(4):611-620; Serafiniet al., Hum Gene Ther, 2004, 14, 63-76; van Meerten et al., Gene Ther,2006, 13, 789-797). Similar systems using EGF-R and Cetuximab orPanitumumab are described in International PCT Publication No. WO2018006880. Additional safety-switch systems include transgenes encodingpro-apoptotic molecules comprising one or more binding sites for achemical inducer of dimerization (CID), enabling elimination of modifiedimmune effector cells by administration of a CID which inducesoligomerization of the pro-apoptotic molecules and activation of theapoptosis pathway. In some embodiments, the pro-apoptotic molecule isFas (also known as CD95) (Thomis et al., Blood, 2001, 97(5), 1249-1257).In some embodiments, the pro-apoptotic molecule is caspase-9 (Straathofet al., Blood, 2005, 105(11), 4247-4254).

In some embodiments, the modified immune effector cells described hereinfurther comprise an exogenous transgene encoding a chimeric switchreceptor. Chimeric switch receptors are engineered cell-surfacereceptors comprising an extracellular domain from an endogenouscell-surface receptor and a heterologous intracellular signaling domain,such that ligand recognition by the extracellular domain results inactivation of a different signaling cascade than that activated by thewild type form of the cell-surface receptor. In some embodiments, thechimeric switch receptor comprises the extracellular domain of aninhibitory cell-surface receptor fused to an intracellular domain thatleads to the transmission of an activating signal rather than theinhibitory signal normally transduced by the inhibitory cell-surfacereceptor. In particular embodiments, extracellular domains derived fromcell-surface receptors known to inhibit immune effector cell activationcan be fused to activating intracellular domains. Engagement of thecorresponding ligand will then activate signaling cascades thatincrease, rather than inhibit, the activation of the immune effectorcell. For example, in some embodiments, the modified immune effectorcells described herein comprise a transgene encoding a PD1-CD28 switchreceptor, wherein the extracellular domain of PD1 is fused to theintracellular signaling domain of CD28 (See e.g., Liu et al., Cancer Res76:6 (2016), 1578-1590 and Moon et al., Molecular Therapy 22 (2014),S201). In some embodiments, the modified immune effector cells describedherein comprise a transgene encoding the extracellular domain of CD200Rand the intracellular signaling domain of CD28 (See Oda et al., Blood130:22 (2017), 2410-2419).

In some embodiments, the modified immune effector cells described hereinfurther comprise an engineered antigen-specific receptor recognizing aprotein target expressed by a target cell, such as a tumor cell or anantigen presenting cell (APC), referred to herein as “modifiedreceptor-engineered cells” or “modified RE-cells”. The term “engineeredantigen receptor” refers to a non-naturally occurring antigen-specificreceptor such as a chimeric antigen receptor (CAR) or a recombinant Tcell receptor (TCR). In some embodiments, the engineered antigenreceptor is a CAR comprising an extracellular antigen binding domainfused via hinge and transmembrane domains to a cytoplasmic domaincomprising a signaling domain. In some embodiments, the CARextracellular domain binds to an antigen expressed by a target cell inan MHC-independent manner leading to activation and proliferation of theRE cell. In some embodiments, the extracellular domain of a CARrecognizes a tag fused to an antibody or antigen-binding fragmentthereof. In such embodiments, the antigen-specificity of the CAR isdependent on the antigen-specificity of the labeled antibody, such thata single CAR construct can be used to target multiple different antigensby substituting one antibody for another (See e.g., U.S. Pat. Nos.9,233,125 and 9,624,279; US Patent Application Publication Nos.20150238631 and 20180104354). In some embodiments, the extracellulardomain of a CAR may comprise an antigen binding fragment derived from anantibody. Antigen binding domains that are useful in the presentdisclosure include, for example, scFvs; antibodies; antigen bindingregions of antibodies; variable regions of the heavy/light chains; andsingle chain antibodies.

In some embodiments, the intracellular signaling domain of a CAR may bederived from the TCR complex zeta chain (such as CD3ξ signalingdomains), FcγRIII, FcεRI, or the T-lymphocyte activation domain. In someembodiments, the intracellular signaling domain of a CAR furthercomprises a costimulatory domain, for example a 4-1BB, CD28, CD40,MyD88, or CD70 domain. In some embodiments, the intracellular signalingdomain of a CAR comprises two costimulatory domains, for example any twoof 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structuresand intracellular signaling domains are known in the art (See e.g., WO2009/091826; US 20130287748; WO 2015/142675; WO 2014/055657; and WO2015/090229, incorporated herein by reference).

CARs specific for a variety of tumor antigens are known in the art, forexample CD171-specific CARs (Park et al., Mol Ther (2007)15(4):825-833), EGFRvIII-specific CARs (Morgan et al., Hum Gene Ther(2012) 23(10):1043-1053), EGF-R-specific CARs (Kobold et al., J NatlCancer Inst (2014) 107(1):364), carbonic anhydrase K-specific CARs(Lamers et al., Biochem Soc Trans (2016) 44(3):951-959), FR-α-specificCARs (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015),HER2-specific CARs (Ahmed et al., J Clin Oncol (2015) 33(15) 1688-1696;Nakazawa et al., Mol Ther (2011) 19(12):2133-2143; Ahmed et al., MolTher (2009) 17(10):1779-1787; Luo et al., Cell Res (2016) 26(7):850-853;Morgan et al., Mol Ther (2010) 18(4):843-851; Grada et al., Mol TherNucleic Acids (2013) 9(2):32), CEA-specific CARs (Katz et al., ClinCancer Res (2015) 21(14):3149-3159), IL13Ra2-specific CARs (Brown etal., Clin Cancer Res (2015) 21(18):4062-4072), GD2-specific CARs (Louiset al., Blood (2011) 118(23):6050-6056; Caruana et al., Nat Med (2015)21(5):524-529), ErbB2-specific CARs (Wilkie et al., J Clin Immunol(2012) 32(5):1059-1070), VEGF-R-specific CARs (Chinnasamy et al., CancerRes (2016) 22(2):436-447), FAP-specific CARs (Wang et al., CancerImmunol Res (2014) 2(2):154-166), MSLN-specific CARs (Moon et al, ClinCancer Res (2011) 17(14):4719-30), NKG2D-specific CARs (VanSeggelen etal., Mol Ther (2015) 23(10):1600-1610), CD19-specific CARs (Axicabtageneciloleucel (Yescarte) and Tisagenlecleucel (Kymriah®) See also, Li etal., J Hematol and Oncol (2018) 11(22), reviewing clinical trials oftumor-specific CARs.

In some embodiments, the engineered antigen receptor is an engineeredTCR. Engineered TCRs comprise TCRα and/or TCRβ chains that have beenisolated and cloned from T cell populations recognizing a particulartarget antigen. For example, TCRα and/or TCRβ genes (i.e., TRAC andTRBC) can be cloned from T cell populations isolated from individualswith particular malignancies or T cell populations that have beenisolated from humanized mice immunized with specific tumor antigens ortumor cells. Engineered TCRs recognize antigen through the samemechanisms as their endogenous counterparts (e.g., by recognition oftheir cognate antigen presented in the context of majorhistocompatibility complex (MHC) proteins expressed on the surface of atarget cell). This antigen engagement stimulates endogenous signaltransduction pathways leading to activation and proliferation of theTCR-engineered cells.

Engineered TCRs specific for tumor antigens are known in the art, forexample WT1-specific TCRs (JTCR016, Juno Therapeutics; WT1-TCRc4,described in US Patent Application Publication No. 20160083449), MART-1specific TCRs (including the DMF4T clone, described in Morgan et al.,Science 314 (2006) 126-129); the DMFST clone, described in Johnson etal., Blood 114 (2009) 535-546); and the ID3T clone, described in van denBerg et al., Mol. Ther. 23 (2015) 1541-1550), gp100-specific TCRs(Johnson et al., Blood 114 (2009) 535-546), CEA-specific TCRs (Parkhurstet al., Mol Ther. 19 (2011) 620-626), NY-ESO and LAGE-1 specific TCRs(1G4T clone, described in Robbins et al., J Clin Oncol 26 (2011)917-924; Robbins et al., Clin Cancer Res 21 (2015) 1019-1027; andRapoport et al., Nature Medicine 21 (2015) 914-921), andMAGE-A3-specific TCRs (Morgan et al., J Immunother 36 (2013) 133-151)and Linette et al., Blood 122 (2013) 227-242). (See also, Debets et al.,Seminars in Immunology 23 (2016) 10-21).

In some embodiments, the engineered antigen receptor is directed againsta target antigen selected from a cluster of differentiation molecule,such as CD3, CD4, CD8, CD16, CD24, CD25, CD33, CD34, CD45, CD64, CD71,CD78, CD80 (also known as B7-1), CD86 (also known as B7-2), CD96, CD116,CD117, CD123, CD133, and CD138, CD371 (also known as CLL1); atumor-associated surface antigen, such as 5T4, BCMA (also known as CD269and TNFRSF17, UniProt #Q02223), carcinoembryonic antigen (CEA), carbonicanhydrase 9 (CAIX or MN/CAIX), CD19, CD20, CD22, CD30, CD40,disialogangliosides such as GD2, ELF2M, ductal-epithelial mucin, ephrinB2, epithelial cell adhesion molecule (EpCAM), ErbB2 (HER2/neu), FCRLS(UniProt #Q68SN8), FKBP11 (UniProt #Q9NYL4), glioma-associated antigen,glycosphingolipids, gp36, GPRCSD (UniProt #Q9NZD1), mut hsp70-2,intestinal carboxyl esterase, IGF-I receptor, ITGA8 (UniProt #P53708),KAMP3, LAGE-la, MAGE, mesothelin, neutrophil elastase, NKG2D, Nkp30,NY-ESO-1, PAP, prostase, prostate-carcinoma tumor antigen-1 (PCTA-1),prostate specific antigen (PSA), PSMA, prostein, RAGE-1, ROR1, RU1(SFMBT1), RU2 (DCDC2), SLAMF7 (UniProt #Q9NQ25), survivin, TAG-72, andtelomerase; a major histocompatibility complex (MHC) molecule presentinga tumor-specific peptide epitope; tumor stromal antigens, such as theextra domain A (EDA) and extra domain B (EDB) of fibronectin; the A1domain of tenascin-C (TnC A1) and fibroblast associated protein (FAP);cytokine receptors, such as epidermal growth factor receptor (EGFR),EGFR variant III (EGFRvIII), TFGβ-R or components thereof such asendoglin; a major histocompatibility complex (MHC) molecule; avirus-specific surface antigen such as an HIV-specific antigen (such asHIV gp120); an EBV-specific antigen, a CMV-specific antigen, aHPV-specific antigen, a Lassa virus-specific antigen, an Influenzavirus-specific antigen as well as any derivate or variant of thesesurface antigens.

A. Effector Functions

In some embodiments, the modified immune effector cells described hereindemonstrate an increase in one or more immune cell effector functions.Herein, the term “effector function” refers to functions of an immunecell related to the generation, maintenance, and/or enhancement of animmune response against a target cell or target antigen. In someembodiments, the modified immune effector cells described hereindemonstrate one or more of the following characteristics compared to anunmodified immune effector cell: increased infiltration or migration into a tumor, increased proliferation, increased or prolonged cellviability, increased resistance to inhibitory factors in the surroundingmicroenvironment such that the activation state of the cell is prolongedor increased, increased production of pro-inflammatory immune factors(e.g., pro-inflammatory cytokines, chemokines, and/or enzymes),increased cytotoxicity, and/or increased resistance to exhaustion.

In some embodiments, the modified immune effector cells described hereindemonstrate increased infiltration into a tumor compared to anunmodified immune effector cell. In some embodiments, increased tumorinfiltration by modified immune effector cells refers to an increase thenumber of modified immune effector cells infiltrating into a tumorduring a given period of time compared to the number of unmodifiedimmune effector cells that infiltrate into a tumor during the sameperiod of time. In some embodiments, the modified immune effector cellsdemonstrate a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, or more fold increase in tumor filtration compared to anunmodified immune cell. Tumor infiltration can be measured by isolatingone or more tumors from a subject and assessing the number of modifiedimmune cells in the sample by flow cytometry, immunohistochemistry,and/or immunofluorescence.

In some embodiments, the modified immune effector cells described hereindemonstrate an increase in cell proliferation compared to an unmodifiedimmune effector cell. In these embodiments, the result is an increase inthe number of modified immune effector cells present compared tounmodified immune effector cells after a given period of time. Forexample, in some embodiments, modified immune effector cells demonstrateincreased rates of proliferation compared to unmodified immune effectorcells, wherein the modified immune effector cells divide at a more rapidrate than unmodified immune effector cells. In some embodiments, themodified immune effector cells demonstrate a 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more fold increase inthe rate of proliferation compared to an unmodified immune cell. In someembodiments, modified immune effector cells demonstrate prolongedperiods of proliferation compared to unmodified immune effector cells,wherein the modified immune effector cells and unmodified immuneeffector cells divide at similar rates, but wherein the modified immuneeffector cells maintain the proliferative state for a longer period oftime. In some embodiments, the modified immune effector cells maintain aproliferative state for 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, or more times longer than an unmodified immunecell.

In some embodiments, the modified immune effector cells described hereindemonstrate increased or prolonged cell viability compared to anunmodified immune effector cell. In such embodiments, the result is anincrease in the number of modified immune effector cells or presentcompared to unmodified immune effector cells after a given period oftime. For example, in some embodiments, modified immune effector cellsdescribed herein remain viable and persist for 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more times longer thanan unmodified immune cell.

In some embodiments, the modified immune effector cells described hereindemonstrate increased resistance to inhibitory factors compared to anunmodified immune effector cell. Exemplary inhibitory factors includesignaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3,IDO) and/or inhibitory cytokines (e.g., IL-10, TGFβ).

In some embodiments, the modified T cells described herein demonstrateincreased resistance to T cell exhaustion compared to an unmodified Tcell. T cell exhaustion is a state of antigen-specific T celldysfunction characterized by decreased effector function and leading tosubsequent deletion of the antigen-specific T cells. In someembodiments, exhausted T cells lack the ability to proliferate inresponse to antigen, demonstrate decreased cytokine production, and/ordemonstrate decreased cytotoxicity against target cells such as tumorcells. In some embodiments, exhausted T cells are identified by alteredexpression of cell surface markers and transcription factors, such asdecreased cell surface expression of CD122 and CD127; increasedexpression of inhibitory cell surface markers such as PD1, LAG3, CD244,CD160, TIM3, and/or CTLA4; and/or increased expression of transcriptionfactors such as Blimp1, NFAT, and/or BATF. In some embodiments,exhausted T cells demonstrate altered sensitivity to cytokine signaling,such as increased sensitivity to TGFβ signaling and/or decreasedsensitivity to IL-7 and IL-15 signaling. T cell exhaustion can bedetermined, for example, by co-culturing the T cells with a populationof target cells and measuring T cell proliferation, cytokine production,and/or lysis of the target cells. In some embodiments, the modifiedimmune effector cells described herein are co-cultured with a populationof target cells (e.g., autologous tumor cells or cell lines that havebeen engineered to express a target tumor antigen) and effector cellproliferation, cytokine production, and/or target cell lysis ismeasured. These results are then compared to the results obtained fromco-culture of target cells with a control population of immune cells(such as unmodified immune effector cells or immune effector cells thathave a control modification).

In some embodiments, resistance to T cell exhaustion is demonstrated byincreased production of one or more cytokines (e.g., IFNγ, TNFα, orIL-2) from the modified immune effector cells compared to the cytokineproduction observed from the control population of immune cells. In someembodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60,70, 80, 90, 100 or more fold increase in cytokine production from themodified immune effector cells compared to the cytokine production fromthe control population of immune cells is indicative of an increasedresistance to T cell exhaustion. In some embodiments, resistance to Tcell exhaustion is demonstrated by increased proliferation of themodified immune effector cells compared to the proliferation observedfrom the control population of immune cells. In some embodiments, a 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5,6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or morefold increase in proliferation of the modified immune effector cellscompared to the proliferation of the control population of immune cellsis indicative of an increased resistance to T cell exhaustion. In someembodiments, resistance to T cell exhaustion is demonstrated byincreased target cell lysis by the modified immune effector cellscompared to the target cell lysis observed by the control population ofimmune cells. In some embodiments, a 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30,35, 40, 45, 50, 60, 70, 80, 90, 100 or more fold increase in target celllysis by the modified immune effector cells compared to the target celllysis by the control population of immune cells is indicative of anincreased resistance to T cell exhaustion.

In some embodiments, exhaustion of the modified immune effector cellscompared to control populations of immune cells is measured during thein vitro or ex vivo manufacturing process. For example, in someembodiments, TILs isolated from tumor fragments are modified accordingto the methods described herein and then expanded in one or more roundsof expansion to produce a population of modified TILs. In suchembodiments, the exhaustion of the modified TILs can be determinedimmediately after harvest and prior to a first round of expansion, afterthe first round of expansion but prior to a second round of expansion,and/or after the first and the second round of expansion. In someembodiments, exhaustion of the modified immune effector cells comparedto control populations of immune cells is measured at one or more timepoints after transfer of the modified immune effector cells into asubject. For example, in some embodiments, the modified cells areproduced according to the methods described herein and administered to asubject. Samples can then be taken from the subject at various timepoints after the transfer to determine exhaustion of the modified immuneeffector cells in vivo over time.

In some embodiments, the modified immune effector cells described hereindemonstrate increased expression or production of pro-inflammatoryimmune factors compared to an unmodified immune effector cell. Examplesof pro-inflammatory immune factors include cytolytic factors, such asgranzyme B, perforin, and granulysin; and pro-inflammatory cytokinessuch as interferons (IFNα, IFNβ, IFNγ), TNFα, IL-1β, IL-12, IL-2, IL-17,CXCL8, and/or IL-6.

In some embodiments, the modified immune effector cells described hereindemonstrate increased cytotoxicity against a target cell compared to anunmodified immune effector cell. In some embodiments, the modifiedimmune effector cells demonstrate a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or more fold increase in cytotoxicityagainst a target cell compared to an unmodified immune cell.

Assays for measuring immune effector function are known in the art. Forexample, tumor infiltration can be measured by isolating tumors from asubject and determining the total number and/or phenotype of thelymphocytes present in the tumor by flow cytometry,immunohistochemistry, and/or immunofluorescence. Cell-surface receptorexpression can be determined by flow cytometry, immunohistochemistry,immunofluorescence, Western blot, and/or qPCR. Cytokine and chemokineexpression and production can be measured by flow cytometry,immunohistochemistry, immunofluorescence, Western blot, ELISA, and/orqPCR. Responsiveness or sensitivity to extracellular stimuli (e.g.,cytokines, inhibitory ligands, or antigen) can be measured by assayingcellular proliferation and/or activation of downstream signalingpathways (e.g., phosphorylation of downstream signaling intermediates)in response to the stimuli. Cytotoxicity can be measured by target-celllysis assays known in the art, including in vitro or ex vivo co-cultureof the modified immune effector cells with target cells and in vivomurine tumor models, such as those described throughout the Examples.

B. Regulation of Endogenous Pathways and Genes

In some embodiments, the modified immune effector cells described hereindemonstrate a reduced expression or function of one or more endogenoustarget genes and/or comprise a gene-regulating system capable ofreducing the expression and/or function of one or more endogenous targetgenes (described infra). In some embodiments, the one or more endogenoustarget genes are present in pathways related to the activation andregulation of effector cell responses. In such embodiments, the reducedexpression or function of the one or more endogenous target genesenhances one or more effector functions of the immune cell.

Exemplary pathways suitable for regulation by the methods describedherein are shown in Table 1. In some embodiments, the expression of anendogenous target gene in a particular pathway is reduced in themodified immune effector cells. In some embodiments, the expression of aplurality (e.g., two or more) of endogenous target genes in a particularpathway are reduced in the modified immune effector cells. For example,the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endogenous targetgenes in a particular pathway may be reduced. In some embodiments, theexpression of an endogenous target gene in one pathway and theexpression of an endogenous target genes in another pathway is reducedin the modified immune effector cells. In some embodiments, theexpression of a plurality of endogenous target genes in one pathway andthe expression of a plurality of endogenous target genes in anotherpathway are reduced in the modified immune effector cells. For example,the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more endogenous targetgenes in one pathway may be reduced and the expression of 2, 3, 4, 5, 6,7, 8, 9, 10, or more endogenous target genes in another particularpathway may be reduced.

In some embodiments, the expression of a plurality of endogenous targetgenes in a plurality of pathways is reduced. For example, one endogenousgene from each of a plurality of pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, or more pathways) may be reduced. In additional aspects, a pluralityof endogenous genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes)from each of a plurality of pathways (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,or more pathways) may be reduced.

TABLE 1 Exemplary Endogenous Pathways Pathway Description LymphocyteSignaling pathway which controls stem cell differentiationdifferentiation from a common lymphoid progenitor to the distinctivelymphocyte type (T cell, B cell or NK cell) NFκβ Signaling pathway thatcontrols transcription of DNA, signaling cytokine production and cellsurvival generally in response to harmful cell stimuli. TGF-β Signalingpathway that regulates cell growth, cell signaling differentiation,apoptosis, cellular homeostasis and other cellular functions. T cellPathway that is initiated by binding of the T cell receptor activation(TCR) complex to a major histocompatibility complex molecule carrying apeptide antigen and by binding of the co- stimulatory receptor CD28 toproteins in the surface of the antigen presenting cell. Activation of aTCR initiates a signaling pathway which triggers antibody production,activation of phagocytic cells and direct cell killing. T cell Signalingpathway that controls programmed cell death in growth response to eitherextrinsic signals or intrinsic cellular stresses Pyrimidine A de novonucleotide biosynthesis pathway for components of biosynthesis RNA andDNA Cytokine Signaling pathways down stream of cytokine receptors,Signaling typically involve positive JAK/STAT signaling Apoptosis Genesthat initiate either the intrinsic or extrinsic apoptotic initiationpathway, which drives programed cell death of the cell TranscriptionGenes that directly bind the promoters of target genes and actinitiation as repressors or transcriptional activators of target genetranscription Ca2++ binding Ca2++ serves as a second messenger inresponse to stimuli and drives intracellular signaling in a number ofprocesses, including inflammation and the immune response. In T cells,Ca2++ signaling is required for the activation of T cells in response toantigen

Exemplary endogenous target genes are shown below in Tables 2 and 3.

In some embodiments, the modified effector cells comprise reducedexpression and/or function of one or more of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAGS, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR (e.g., one or more endogenous target genes selected fromTable 2). In some embodiments, the modified effector cells comprisereduced expression and/or function of one or more of TNFAIP3, CBLB, orBCOR.

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of at least two genes selected from IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, and BCOR (e.g., at least two genes selected fromTable 2). For example, in some embodiments, the modified immune effectorcells comprise reduced expression and/or function of at least two genesselected from Combination Nos. 1-600, as illustrated in FIG. 1A-FIG. 1B.In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of BCOR and reduced expression and/orfunction of CBLB. While exemplary methods for modifying the expressionof IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and/or BCOR are described herein, theexpression of these endogenous target genes may also be modified bymethods known in the art. For example, inhibitory antibodies against PD1(encoded by PDCD1), NRP1, HACR2, LAG3, TIGIT, and CTLA4 are known in theart and some are FDA approved for oncologic indications (e.g., nivolumaband pembrolizumab for PD1).

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of one or more of BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, WDR6, E2F8, SERPINA3, GNAS,ZC3H12A, MAP4K1, and NR4A3 (e.g., one or more endogenous target genesselected from Table 3).

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Semaphorin 7A,(SEMA7A) gene, also known as CD108. In some embodiments, the modifiedeffector cells described herein comprise an inactivating mutation in theSEMA7A gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the RNA-binding protein39 (RBM39) gene. The RBM39 protein is found in the nucleus, where itcolocalizes with core spliceosomal proteins. Studies of a mouse proteinwith high sequence similarity to this protein suggest that this proteinmay act as a transcriptional coactivator for JUN/AP-1 and estrogenreceptors. In some embodiments, the modified effector cells describedherein comprise an inactivating mutation in the RBM39 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Bcl-2-like protein 11(BCL2L11) gene, also commonly called BIM. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the BCL2L11 gene

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Friend leukemiaintegration 1 transcription factor (FLI1) gene, also known astranscription factor ERGB. In some embodiments, the modified effectorcells described herein comprise an inactivating mutation in the FLI1gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Calmodulin 2 (CALM2)gene. In some embodiments, the modified effector cells described hereincomprise an inactivating mutation in the CALM2 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Dihydroorotatedehydrogenase gene (DHODH) gene. The DHODH protein is a mitochondrialprotein located on the outer surface of the inner mitochondrial membraneand catalyzes the ubiquinone-mediated oxidation of dihydroorotate toorotate in de novo pyrimidine biosynthesis. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the DHODH gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the uridine monophosphatesynthase (UMPS) gene, also referred to as orotate phosphoribosyltransferase or orotidine-5′-decarboxylase. The UMPS protein catalysesthe formation of uridine monophosphate (UMP), an energy-carryingmolecule in many important biosynthetic pathways. In some embodiments,the modified effector cells described herein comprise an inactivatingmutation in the UMPS gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the cysteine richhydrophobic domain 2 (CHIC2) gene. The encoded CHIC2 protein contains acysteine-rich hydrophobic (CHIC) motif, and is localized to vesicularstructures and the plasma membrane and is associated with some cases ofacute myeloid leukemia. In some embodiments, the modified effector cellsdescribed herein comprise an inactivating mutation in the CHIC2 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Poly(rC)-bindingprotein 1 (PCBP1) gene. In some embodiments, the modified effector cellsdescribed herein comprise an inactivating mutation in the PCBP1 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the Protein polybromo-1(PBRM1) gene, also known as BRG1-associated factor 180 (BAF180). PBRM1is a component of the SWI/SNF-B chromatin-remodeling complex, and is atumor suppressor gene in many cancer subtypes. Mutations are especiallyprevalent in clear cell renal cell carcinoma. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the PBRM1 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the WD repeat-containingprotein 6 (WDR6) gene, a member of the WD repeat protein familyubiquitously expressed in adult and fetal tissues. WD repeats areminimally conserved regions of approximately 40 amino acids typicallybracketed by gly-his and trp-asp (GH-WD), which may facilitate formationof heterotrimeric or multiprotein complexes. Members of this family areinvolved in a variety of cellular processes, including cell cycleprogression, signal transduction, apoptosis, and gene regulation. Insome embodiments, the modified effector cells described herein comprisean inactivating mutation in the WDR6 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the E2F transcriptionfactor 8 (E2F8) gene. The encoded E2F8 protein regulates progressionfrom G1 to S phase by ensuring the nucleus divides at the proper time.In some embodiments, the modified effector cells described hereincomprise an inactivating mutation in the E2F8 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the serpin family Amember 3 (SERPINA3) gene. SERPINA3 encodes the Alpha 1-antichymotrypsin(α1AC, A1AC, or a1ACT) protein, which inhibits the activity of certainproteases, such as cathepsin G and chymases. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the SERPINA3 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the GNAS complex locus(GNAS) gene. It is the stimulatory G-protein alpha subunit (Gs-α), a keycomponent of many signal transduction pathways. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the GNAS gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the ZC3H12A gene.Zc3h12a, also referred to as MCPIP1 and Regnase-1, is a RNase thatpossesses a RNase domain just upstream of a CCCH-type zinc-finger motif.Through its nuclease activity, Zc3h12a targets and destabilizes themRNAs of transcripts, such as IL-6, by binding a conserved stem loopstructure within the 3′ UTR of these genes. In T cells, Zc3h12a controlsthe transcript levels of a number of pro-inflammatory genes, includingc-Rel, Ox40, and IL-2. In monocytes, Zc3h12a downregulates IL-6 andIL-12B mRNAs, thus mitigating inflammation. In cancer cells, Zc3h12apromotes apoptosis by inhibiting anti-apoptotic genes including Bcl2L1,Bcl2A1, RelB and Bcl3. Zc3h12a activation is transient and is subject tonegative feedback mechanisms including proteasome-mediated degradationor mucosa-associated lymphoid tissue 1 (MALT1) mediated cleavage. Insome embodiments, the modified effector cells described herein comprisean inactivating mutation in the ZC3H12A gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the MAP4K1 gene. MAP4K1,also referred to as HPK1, is a member of the MAP4K family of mammalianSte20-related protein kinases and is a protein serine-threonine kinasepredominantly expressed in hematopoietic organs. The MAP4K1 proteincomprises an N-terminal kinase domain followed by four proline-richmotifs capable of binding to proteins containing Src homology 3 domains.Upon phosphorylation by kinases such as Lck and Zap70 and in thepresence of ATP, MAP4K1 possesses catalytic activity and mediatesautophosphorylation as well as phosphorylation of downstream substrateproteins such as SLP-76 and MAP3K proteins. Phosphorylation of SLP-76 atSer-376 leads to both the ubiquitination and proteosomal degradation ofSLP-76 as well as increased interactions between SLP-76 and 14-3-3τ,which negatively regulates TCR signaling. In some embodiments, themodified effector cells described herein comprise an inactivatingmutation in the MAP4K1 gene.

In some embodiments, the modified effector cells described hereincomprise reduced expression and/or function of the NR4A3 gene. NR4A3 isan inducible orphan receptor and member of the steroid-thyroidhormone-retinoid receptor superfamily, and is a transcriptionalactivator. When expression is induced by various stimuli, NR4A3 drivesgene expression in a ligand-independent way by translocating from thecytoplasm to the nucleus and binding to NR4A1 response elements (NBRE)as monomers and Nur response elements (NurRE) as homodimers. NR4A3 thendrives the transcription of discrete sets of genes controlling cellularsurvival and differentiation both within and outside the immune system.In some embodiments, the modified effector cells described hereincomprise an inactivating mutation in the NR4A3 gene.

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of at least two genes selected from BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., two or moregenes selected from Table 3). For example, in some embodiments, themodified immune effector cells comprise reduced expression and/orfunction of at least two genes selected from Combination Nos. 1176-1681,as illustrated in FIG. 3A-FIG. 3B. In some embodiments, the modifiedimmune effector cells comprise reduced expression and/or function of atleast two genes selected from Combination Nos. 1176-1483, as illustratedin FIG. 3A. In some embodiments, the modified immune effector cellscomprise reduced expression and/or function of at least two genesselected from Combination Nos. 1250-1265, as illustrated in FIG. 3B. Insome embodiments, the modified immune effector cells comprise reducedexpression and/or function of at least two genes selected fromCombination Nos. 1266-1281, as illustrated in FIG. 3B. In someembodiments, the modified immune effector cells comprise reducedexpression and/or function of at least two genes selected fromCombination Nos. 1282-1297, as illustrated in FIG. 3B.

In some embodiments, the modified effector cells comprise reducedexpression and/or function of one or more of BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., one or more gene selected fromTable 3) and one or more of IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR (e.g.,one or more gene selected from Table 2). For example, the modifiedimmune effector cells may comprise reduced expression and/or function ofa combination of an endogenous target genes selected from CombinationNos. 601-1025. In some embodiments, the modified immune effector cellsmay comprise reduced expression and/or function of a combination of twoendogenous target genes selected from Combination Nos. 601-950 (asillustrated in FIG. 2A). In some embodiments, the modified immuneeffector cells may comprise reduced expression and/or function of acombination of two endogenous target genes selected from CombinationNos. 951-975 (as illustrated in FIG. 2B). In some embodiments, themodified immune effector cells may comprise reduced expression and/orfunction of a combination of two endogenous target genes selected fromCombination Nos. 976-1000 (as illustrated in FIG. 2B). In someembodiments, the modified immune effector cells may comprise reducedexpression and/or function of a combination of two endogenous targetgenes selected from Combination Nos. 1001-1025 (as illustrated in FIG.2B).

In some embodiments, the modified effector cells comprise reducedexpression and/or function of at least one gene selected from BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, and GNAS and reduced expression and/or function of atleast one gene selected from TNFAIP3, CBLB, or BCOR. In someembodiments, the modified effector cells comprise reduced expressionand/or function of ZC3H12A and at least one gene selected from TNFAIP3,CBLB, or BCOR. In some embodiments, the modified effector cells compriseinactivating mutations in ZC3H12A and at least one gene selected fromTNFAIP3, CBLB, or BCOR. In some embodiments, the modified effector cellscomprise reduced expression and/or function of MAP4K1 and at least onegene selected from TNFAIP3, CBLB, or BCOR. In some embodiments, themodified effector cells comprise inactivating mutations in MAP4K1 and atleast one gene selected from TNFAIP3, CBLB, or BCOR. In someembodiments, the modified effector cells comprise reduced expressionand/or function of NR4A3 and at least one gene selected from TNFAIP3,CBLB, or BCOR. In some embodiments, the modified effector cells compriseinactivating mutations in NR4A3 and at least one gene selected fromTNFAIP3, CBLB, or BCOR.

In some embodiments, the modified effector cells comprise reducedexpression and/or function of at least one gene selected from BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 and reduced expressionand/or function of CBLB. In some embodiments, the modified effectorcells comprise reduced expression and/or function of at least one geneselected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS and reduced expressionand/or function of CBLB. In some embodiments, the modified effectorcells comprise reduced expression and/or function of ZC3H12A and CBLB.In some embodiments, the modified effector cells comprise inactivatingmutations in ZC3H12A and CBLB. In some embodiments, the modifiedeffector cells comprise reduced expression and/or function of MAP4K1 andCBLB. In some embodiments, the modified effector cells compriseinactivating mutations in MAP4K1 and CBLB. In some embodiments, themodified effector cells comprise reduced expression and/or function ofNR4A3 and CBLB. In some embodiments, the modified effector cellscomprise inactivating mutations in NR4A3 and CBLB.

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of a gene selected from IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR (e.g., one or more gene selected from Table 2) andreduced expression and/or function of two genes selected from BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., one or more geneselected from Table 3). For example, in some embodiments, the modifiedimmune effector cells comprises reduced expression and/or function of agene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA,SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D,NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, and BCOR in addition toreduced expression and/or function of two endogenous target genecombinations selected from Combination Nos. 1026-1297 (as illustrated inFIG. 3A-FIG. 3B).

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of a gene selected from BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., a gene selected from Table 3)and reduced expression and/or function of two genes selected from IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT,CTLA4, PTPN6, PDCD1, or BCOR (e.g., one or more gene selected from Table2). For example, in some embodiments, the modified immune effector cellscomprise reduced expression and/or function of any one of BCL2L11, FLI1,CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8,SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 in addition to reducedexpression and/or function of two endogenous target gene combinationsselected from Combination Nos. 1-600 illustrated in FIG. 1A-FIG. 1B. Insome embodiments, the modified immune effector cells comprise reducedexpression and/or function of a gene selected from BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,and GNAS in addition to reduced expression and/or function of twoendogenous target gene combinations selected from Combination Nos. 1-600illustrated in FIG. 1A-FIG. 1B. In some embodiments, the modified immuneeffector cells comprise reduced expression and/or function of ZC3H12A inaddition to reduced expression and/or function of two endogenous targetgene combinations selected from Combination Nos. 1-600 illustrated inFIG. 1A-FIG. 1B. In some embodiments, the modified immune effector cellscomprise reduced expression and/or function of MAP4K1 in addition toreduced expression and/or function of two endogenous target genecombinations selected from Combination Nos. 1-600 illustrated in FIG.1A-FIG. 1B. In some embodiments, the modified immune effector cellscomprise reduced expression and/or function of NR4A3 in addition toreduced expression and/or function of two endogenous target genecombinations selected from Combination Nos. 1-600 illustrated in FIG.1A-FIG. 1B.

In some embodiments, the modified immune effector cells comprise reducedexpression and/or function of a plurality of genes selected from Table 2and reduced expression and/or function of a plurality of genes selectedfrom Table 3. In some embodiments, the modified immune effector cellscomprise reduced expression and/or function of two genes selected fromTable 2 and reduced expression and/or function of two genes selectedfrom Table 3. For example, in some embodiments, the modified immuneeffector cells comprise reduced expression and/or function of acombination of two genes selected from Combination Nos. 1026-1297 asshown in FIG. 3A-FIG. 3B and a combination of two genes selected fromCombination Nos. 1-600 as shown in FIG. 1A-FIG. 1B. In some embodiments,the modified immune effector cells may comprise reduced expressionand/or function of three or more of IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, orBCOR and reduced expression and/or function of three or more of BCL2L11,FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6,E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3.

TABLE 2 Exemplary Endogenous Genes Human Murine Gene UniProt HumanUniProt Murine Symbol Gene Name Ref. NCBI ID Ref. NCBI ID IKZF1 IKAROSfamily zinc Q13422 10320 Q03267 22778 finger 1 IKZF2 IKAROS family zincQ9UKS7 22807 P81183 22779 finger 2 IKZF3 IKAROS family zinc Q9UKT9 22806O08900 22780 finger 3 NFKBIA NFKB inhibitor P25963 4792 Q9Z1E3 18035alpha BCL3 B cell P20749 602 Q9Z2F6 12051 CLL/lymphoma 3 TNIP1 TNFAIP3interacting Q15025 10318 Q9WUU8 57783 protein 1 TNFAIP3 TNF alphainduced P21580 7128 Q60769 21929 protein 3 SMAD2 SMAD family Q15796 4087Q919P9 17126 member 2 TGFBR1 transforming growth P36897 7046 Q6472921812 factor beta receptor 1 TGFBR2 transforming growth P37173 7048Q623212 21813 factor beta receptor 2 TANK TRAF family Q92844 10010P70347 21353 member associated NFKB activator FOXP3 forkhead box P3Q9BZS1 50943 Q99JB6 20371 CBLB Cbl proto-oncogene Q13191 868 Q3TTA7208650 B PPP2R2D protein phosphatase 2 Q66LE6 55844 Q7ZX64 52432regulatory subunit Bdelta NRP1 neuropilin 1 Q14786 8829 P97333 18186HAVCR2 hepatitis A virus Q8TDQ0 84868 Q8VIM0 171285 cellular receptor 2LAG3 lymphocyte P18627 3902 Q61790 16768 activating 3 TIGIT T cellQ495A1 201633 P86176 100043314 immunoreceptor with Ig and ITIM domainsCTLA4 cytotoxic T- P16410 1493 P09793 12477 lymphocyte associatedprotein 4 PTPN6 protein tyrosine P29350 5777 P29351 15170 phosphatase,non- receptor type 6 BCOR BCL6 corepressor Q6W2J9 54880 Q8CGN4 71458GATA3 GATA binding P23771 2625 P23772 14462 protein 3 PDCD1 Programmedcell Q15116 5133 Q02242 18566 death 1 protein RC3H1 Ring finger andQ5TC82 149041 Q4VGL6 381305 CCCH-type domains 1 TRAF6 TNF receptorQ9Y4K3 7186 P70196 22034 associated factor 6

TABLE 3 Exemplary Genes for Novel Regulation Human Murine Gene UniProtHuman UniProt Murine Symbol Gene Name Ref. NCBI ID Ref. NCBI ID SEMA7Asemaphorin 7A O75326 8482 Q9QUR8 20361 RBM39 RNA binding motif proteinQ14498 9584 Q8VH51 170791 39 BCL2L11 BCL2 like 11 O43521 10018 O5491812125 FLI1 Fli-1 proto-oncogene, ETS Q01543 2313 P26323 14247transcription factor CALM2 calmodulin 2 P0P24 805 P0DP30 12314 DHODHdihydroorotate Q02127 1723 O35435 56749 dehydrogenase (quinone) UMPSuridine monophosphate P11172 7372 P13439 22247 synthetase CHIC2 cysteinerich hydrophobic Q9UKJ5 26511 Q9D9G3 74277 domain 2 PCBP1 poly(rC)binding protein 1 Q15365 5093 P60335 23983 PBRM1 polybromo 1 Q86U8655193 Q8BSQ9 66923 WDR6 WD repeat domain 6 Q9NNW5 11180 Q99ME2 83669E2F8 E2F transcription factor 8 A0AVK6 79733 Q58FA4 108961 SERPINA3serpin family A member 3 P01011 12 GNAS guanine nucleotide bindingQ5JWF2 2778 Q6R0H7 14683 protein, alpha stimulating ZC3H12AEndoribonuclease Q5D1E8 80149 Q5D1E7 230738 ZC3H12A MAP4K1mitogen-activated protein Q92918 11184 P70218 26411 kinase kinase kinasekinase 1 NR4A3 Nuclear receptor subfamily Q92570 8013 Q9QZB6 18124 4group A member 3

III. Gene-Regulating Systems

Herein, the term “gene-regulating system” refers to a protein, nucleicacid, or combination thereof that is capable of modifying an endogenoustarget DNA sequence when introduced into a cell, thereby regulating theexpression or function of the encoded gene product. Numerous geneediting systems suitable for use in the methods of the presentdisclosure are known in the art including, but not limited to, shRNAs,siRNAs, zinc-finger nuclease systems, TALEN systems, and CRISPR/Cassystems.

As used herein, “regulate,” when used in reference to the effect of agene-regulating system on an endogenous target gene encompasses anychange in the sequence of the endogenous target gene, any change in theepigenetic state of the endogenous target gene, and/or any change in theexpression or function of the protein encoded by the endogenous targetgene.

In some embodiments, the gene-regulating system may mediate a change inthe sequence of the endogenous target gene, for example, by introducingone or more mutations into the endogenous target sequence, such as byinsertion or deletion of one or more nucleic acids in the endogenoustarget sequence. Exemplary mechanisms that can mediate alterations ofthe endogenous target sequence include, but are not limited to,non-homologous end joining (NHEJ) (e.g., classical or alternative),microhomology-mediated end joining (MMEJ), homology-directed repair(e.g., endogenous donor template mediated), SDSA (synthesis dependentstrand annealing), single strand annealing or single strand invasion.

In some embodiments, the gene-regulating system may mediate a change inthe epigenetic state of the endogenous target sequence. For example, insome embodiments, the gene-regulating system may mediate covalentmodifications of the endogenous target gene DNA (e.g., cytosinemethylation and hydroxymethylation) or of associated histone proteins(e.g. lysine acetylation, lysine and arginine methylation, serine andthreonine phosphorylation, and lysine ubiquitination and sumoylation).

In some embodiments, the gene-regulating system may mediate a change inthe expression of the protein encoded by the endogenous target gene. Insuch embodiments, the gene-regulating system may regulate the expressionof the encoded protein by modifications of the endogenous target DNAsequence, or by acting on the mRNA product encoded by the DNA sequence.In some embodiments, the gene-regulating system may result in theexpression of a modified endogenous protein. In such embodiments, themodifications to the endogenous DNA sequence mediated by thegene-regulating system result in the expression of an endogenous proteindemonstrating a reduced function as compared to the correspondingendogenous protein in an unmodified immune effector cell. In suchembodiments, the expression level of the modified endogenous protein maybe increased, decreased or may be the same, or substantially similar to,the expression level of the corresponding endogenous protein in anunmodified immune cell.

A. Nucleic Acid-Based Gene-Regulating Systems

As used herein, a nucleic acid-based gene-regulating system is a systemcomprising one or more nucleic acid molecules that is capable ofregulating the expression of an endogenous target gene without therequirement for an exogenous protein. In some embodiments, the nucleicacid-based gene-regulating system comprises an RNA interference moleculeor antisense RNA molecule that is complementary to a target nucleic acidsequence.

An “antisense RNA molecule” refers to an RNA molecule, regardless oflength, that is complementary to an mRNA transcript. Antisense RNAmolecules refer to single stranded RNA molecules that can be introducedto a cell, tissue, or subject and result in decreased expression of anendogenous target gene product through mechanisms that do not rely onendogenous gene silencing pathways, but rather rely on RNaseH-mediateddegradation of the target mRNA transcript. In some embodiments, anantisense nucleic acid comprises a modified backbone, for example,phosphorothioate, phosphorodithioate, or others known in the art, or maycomprise non-natural internucleoside linkages. In some embodiments, anantisense nucleic acid can comprise locked nucleic acids (LNA).

“RNA interference molecule” as used herein refers to an RNApolynucleotide that mediates the decreased the expression of anendogenous target gene product by degradation of a target mRNA throughendogenous gene silencing pathways (e.g., Dicer and RNA-inducedsilencing complex (RISC)). Exemplary RNA interference agents includemicro RNAs (also referred to herein as “miRNAs”), short hair-pin RNAs(shRNAs), small interfering RNAs (siRNAs), RNA aptamers, andmorpholinos.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises one or more miRNAs. miRNAs refers to naturally occurring,small non-coding RNA molecules of about 21-25 nucleotides in length.miRNAs are at least partially complementary to one or more target mRNAmolecules. miRNAs can downregulate (e.g., decrease) expression of anendogenous target gene product through translational repression,cleavage of the mRNA, and/or deadenylation.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises one or more shRNAs. shRNAs are single stranded RNA moleculesof about 50-70 nucleotides in length that form stem-loop structures andresult in degradation of complementary mRNA sequences. shRNAs can becloned in plasmids or in non-replicating recombinant viral vectors to beintroduced intracellularly and result in the integration of theshRNA-encoding sequence into the genome. As such, an shRNA can providestable and consistent repression of endogenous target gene translationand expression.

In some embodiments, nucleic acid-based gene-regulating system comprisesone or more siRNAs. siRNAs refer to double stranded RNA moleculestypically about 21-23 nucleotides in length. The siRNA associates with amulti protein complex called the RNA-induced silencing complex (RISC),during which the “passenger” sense strand is enzymatically cleaved. Theantisense “guide” strand contained in the activated RISC then guides theRISC to the corresponding mRNA because of sequence homology and the samenuclease cuts the target mRNA, resulting in specific gene silencing.Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides inlength and has a 2 base overhang at its 3′ end. siRNAs can be introducedto an individual cell and/or culture system and result in thedegradation of target mRNA sequences. siRNAs and shRNAs are furtherdescribed in Fire et al., Nature, 391:19, 1998 and U.S. Pat. Nos.7,732,417; 8,202,846; and 8,383,599.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises one or more morpholinos. “Morpholino” as used herein refers toa modified nucleic acid oligomer wherein standard nucleic acid bases arebound to morpholine rings and are linked through phosphorodiamidatelinkages. Similar to siRNA and shRNA, morpholinos bind to complementarymRNA sequences. However, morpholinos function through steric-inhibitionof mRNA translation and alteration of mRNA splicing rather thantargeting complementary mRNA sequences for degradation.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNAaptamer, or a morpholino) that binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA encoded by a DNA sequence of a target gene selected from IKZF1,IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK,FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAGS, TIGIT,CTLA4, PTPN6, PDCD1, or BCOR (i.e., those listed in Table 2). In someembodiments, the nucleic acid-based gene-regulating system comprises anucleic acid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a RNAsequence encoded by a DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B. Throughout this application,the referenced genomic coordinates are based on genomic annotations inthe GRCh38 (also referred to as hg38) assembly of the human genome fromthe Genome Reference Consortium, available at the National Center forBiotechnology Information website. Tools and methods for convertinggenomic coordinates between one assembly and another are known in theart and can be used to convert the genomic coordinates provided hereinto the corresponding coordinates in another assembly of the humangenome, including conversion to an earlier assembly generated by thesame institution or using the same algorithm (e.g., from GRCh38 toGRCh37), and conversion an assembly generated by a different institutionor algorithm (e.g., from GRCh38 to NCBI33, generated by theInternational Human Genome Sequencing Consortium). Available methods andtools known in the art include, but are not limited to, NCBI GenomeRemapping Service, available at the National Center for BiotechnologyInformation website, UCSC LiftOver, available at the UCSC Genome Browerwebsite, and Assembly Converter, available at the Ensembl.org website.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNAaptamer, or a morpholino) that binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813. In some embodiments, the nucleic acid-based gene-regulatingsystem is capable of reducing the expression and/or function of CBLB,and comprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNAaptamer, or a morpholino) that binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 499-524. In someembodiments, the nucleic acid-based gene-regulating system is capable ofreducing the expression and/or function of BCOR, and comprises a nucleicacid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to an RNAsequence encoded by one of SEQ ID NOs: 708-772 or SEQ ID NOs: 708-764.In some embodiments, the nucleic acid-based gene-regulating system iscapable of reducing the expression and/or function of TNFAIP3, andcomprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNAaptamer, or a morpholino) that binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 348-396 or SEQ ID NOs:348-386. In some embodiments, the nucleic acid-based gene-regulatingsystem comprises an siRNA molecule or an shRNA molecule selected fromthose known in the art, such as the siRNA and shRNA constructs availablefrom commercial suppliers such as Sigma Aldrich, Dharmacon,ThermoFisher, and the like.

In some embodiments, the endogenous target gene is CBLB and the nucleicacid molecule is an shRNA encoded by a nucleic acid sequence selectedfrom SEQ ID NOs: 41-44 (See International PCT Publication No.2018156886) or selected from SEQ ID NOs: 45-53 (See International PCTPublication No. WO 2017120998). In some embodiments, the endogenoustarget gene is CBLB and the nucleic acid molecule is an siRNA comprisinga nucleic acid sequence selected from SEQ ID NOs: 54-63 (SeeInternational PCT Publication No. WO 2018006880) or SEQ ID NOs: 64-73(See International PCT Publication Nos. WO 2018120998 and WO2018137293).

In some embodiments, the endogenous target gene is TNFAIP3 and thenucleic acid molecule is an shRNA encoded by a nucleic acid sequenceselected from SEQ ID NOs: 74-95 (See U.S. Pat. No. 8,324,369). In someembodiments, the endogenous target gene is TNFAIP3 and the nucleic acidmolecule is an siRNA comprising a nucleic acid sequence selected fromSEQ ID NOs: 96-105 (See International PCT Publication No. WO2018006880).

In some embodiments, the endogenous target gene is CTLA4 and the nucleicacid molecule is an shRNA encoded by a nucleic acid sequence selectedfrom SEQ ID NOs: 128-133 (See International PCT Publication No. WO2017120996). In some embodiments, the endogenous target gene is CTLA4and the nucleic acid molecule is an siRNA comprising a nucleic acidsequence selected from SEQ ID NOs: 134-143 (See International PCTPublication Nos. WO2017120996, WO 2017120998, WO 2018137295, and WO2018137293) or SEQ ID NOs: 144-153 (See International PCT PublicationNo. WO 2018006880).

In some embodiments, the endogenous target gene is PDCD1 and the nucleicacid molecule is an shRNA encoded by a nucleic acid sequence selectedfrom SEQ ID NOs: 106-107 (See International PCT Publication Nos. WO2017120996). In some embodiments, the endogenous target gene is PDCD1and the nucleic acid molecule is an siRNA comprising a nucleic acidsequence selected from SEQ ID NOs: 108-117 (See International PCTPublication Nos. WO2017120996, WO 201712998, WO 2018137295, and WO2018137293) or SEQ ID NOs: 118-127 (See International PCT PublicationNo. WO 2018006880).

In some embodiments, the nucleic acid-based gene-regulating systemcomprises a nucleic acid molecule (e.g., an siRNA, an shRNA, an RNAaptamer, or a morpholino) that binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by a DNA sequence of a target gene selected fromBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (i.e., thoselisted in Table 3). In some embodiments, the nucleic acid-basedgene-regulating system comprises a nucleic acid molecule (e.g., ansiRNA, an shRNA, an RNA aptamer, or a morpholino) that binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or is100% identical to an RNA sequence encoded by a DNA sequence defined by aset of genomic coordinates shown in Table 6A-Table 6H. In someembodiments, the nucleic acid-based gene-regulating system comprises anucleic acid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99%, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 814-1566.

In some embodiments, the nucleic acid-based gene-regulating system iscapable of reducing the expression and/or function of a target geneselected from BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, thenucleic acid-based gene-regulating system comprises a nucleic acidmolecule (e.g., an siRNA, an shRNA, an RNA aptamer, or a morpholino)that binds to a target RNA sequence that is at least 90%, 95%, 96%, 97%,98%, or 99%, or is 100% identical to an RNA sequence encoded by a DNAsequence defined by a set of genomic coordinates shown in one of Table6A or Table 6B. In some embodiments, the nucleic acid-basedgene-regulating system comprises a nucleic acid molecule (e.g., ansiRNA, an shRNA, an RNA aptamer, or a morpholino) that binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or is100% identical to an RNA sequence encoded by one of SEQ ID NOs:814-1064.

In some embodiments, the nucleic acid-based gene-regulating system iscapable of reducing the expression and/or function of ZC3H12A. In someembodiments, the nucleic acid-based gene-regulating system comprises anucleic acid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99%, or is 100% identical to an RNA sequenceencoded by a DNA sequence defined by a set of genomic coordinates shownin one of Table 6C or Table 6D. In some embodiments, the nucleicacid-based gene-regulating system comprises a nucleic acid molecule(e.g., an siRNA, an shRNA, an RNA aptamer, or a morpholino) that bindsto a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99%, or is 100% identical to an RNA sequence encoded by one of SEQ IDNOs: 1065-1509. In some embodiments, the nucleic acid-basedgene-regulating system comprises a nucleic acid molecule (e.g., ansiRNA, an shRNA, an RNA aptamer, or a morpholino) that binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%, or is100% identical to an RNA sequence encoded by one of SEQ ID NOs:1065-1264.

In some embodiments, the nucleic acid-based gene-regulating system iscapable of reducing the expression and/or function of MAP4K1. In someembodiments, the nucleic acid-based gene-regulating system comprises anucleic acid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99%, or is 100% identical to an RNA sequenceencoded by a DNA sequence defined by a set of genomic coordinates shownin one of Table 6E or Table 6F. In some embodiments, the nucleicacid-based gene-regulating system comprises a nucleic acid molecule(e.g., an siRNA, an shRNA, an RNA aptamer, or a morpholino) that bindsto a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99%, or is 100% identical to an RNA sequence encoded by one of SEQ IDNOs: 510-1538.

In some embodiments, the nucleic acid-based gene-regulating system iscapable of reducing the expression and/or function of NR4A3. In someembodiments, the nucleic acid-based gene-regulating system comprises anucleic acid molecule (e.g., an siRNA, an shRNA, an RNA aptamer, or amorpholino) that binds to a target RNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99%, or is 100% identical to an RNA sequenceencoded by a DNA sequence defined by a set of genomic coordinates shownin one of Table 6G or Table 6H. In some embodiments, the nucleicacid-based gene-regulating system comprises a nucleic acid molecule(e.g., an siRNA, an shRNA, an RNA aptamer, or a morpholino) that bindsto a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99%, or is 100% identical to an RNA sequence encoded by one of SEQ IDNOs: 1539-1566.

In some embodiments, the nucleic acid-based gene-regulating systemcomprises an siRNA molecule or an shRNA molecule selected from thoseknown in the art, such as those available from commercial suppliers suchas Sigma Aldrich, Dharmacon, ThermoFisher, and the like. Exemplary siRNAand shRNA constructs are described in Table 4A and Table 4B below. Insome embodiments, the nucleic acid-based gene-regulating systemcomprises two or more siRNA molecules selected from those known in theart, such as the siRNA constructs described in Table 4A. In someembodiments, the nucleic acid-based gene-regulating system comprises twoor more shRNA molecules selected from those known in the art, such asthe shRNA constructs described in Table 4B.

TABLE 4A Exemplary siRNA constructs Target Gene siRNA construct SEMA7AMISSION ® esiRNA human SEMA7A (esiRNA1) (SigmaAldrich Product#EHU143161) MISSION ® esiRNA targeting mouse Sema7a (esiRNA1)(SigmaAldrich Product# EMU010311) human Rosetta Predictions(SigmaAldrich Product# NM_003612) murine Rosetta Predictions(SigmaAldrich Product# NM_011352) RBM39 MISSION ® esiRNA human RBM39(esiRNA1) (SigmaAldrich Product# EHU070351) human Rosetta Predictions(SigmaAldrich Product# NM_004902) human Rosetta Predictions(SigmaAldrich Product# NM_184234) human Rosetta Predictions(SigmaAldrich Product# NM_184237) human Rosetta Predictions(SigmaAldrich Product# NM_184241) human Rosetta Predictions(SigmaAldrich Product# NM_184244) BCL2L11 MISSION ® esiRNA targetingmouse Bcl2l11 (esiRNA1) (SigmaAldrich Product# human Rosetta Predictions(SigmaAldrich Product# NM_006538) human Rosetta Predictions(SigmaAldrich Product# NM_138621) human Rosetta Predictions(SigmaAldrich Product# NM_138622) human Rosetta Predictions(SigmaAldrich Product# NM_138623) human Rosetta Predictions(SigmaAldrich Product# NM_138624) FLI1 MISSION ® esiRNA human FLI1(esiRNA1) (SigmaAldrich Product# EHU091961) MISSION ® esiRNA targetingmouse Fli1 (esiRNA1) (SigmaAldrich Product# EMU090601) human RosettaPredictions (SigmaAldrich Product# NM_002017) murine Rosetta Predictions(SigmaAldrich Product# NM_008026) CALM2 MISSION ® esiRNA human CALM2(esiRNA1) (SigmaAldrich Product# EHU110161) MISSION ® esiRNA targetingmouse Calm2 (SigmaAldrich Product# EMU176331) human Rosetta Predictions(SigmaAldrich Product# NM_001743) murine Rosetta Predictions(SigmaAldrich Product# NM_007589) DHODH MISSION ® esiRNA human DHODH(esiRNA1) (SigmaAldrich Product# EHU138421) MISSION ® esiRNA targetingmouse Dhodh (esiRNA1) (SigmaAldrich Product# EMU072221) human RosettaPredictions (SigmaAldrich Product# NM_001025193) human RosettaPredictions (SigmaAldrich Product# NM_001361) DHODH murine RosettaPredictions (SigmaAldrich Product# NM_020046) UMPS MISSION ® esiRNAhuman UMPS (esiRNA1) (SigmaAldrich Product# EHU093891) MISSION ® esiRNAtargeting mouse Umps (esiRNA1) (SigmaAldrich Product# EMU023181) humanRosetta Predictions (SigmaAldrich Product# NM_000373) murine RosettaPredictions (SigmaAldrich Product# NM_009471) CHIC2 MISSION ® esiRNAhuman CHIC2 (esiRNA1) (SigmaAldrich Product# EHU137501) MISSION ® esiRNAtargeting mouse Chic2 (esiRNA1) (SigmaAldrich Product# EMU019221 humanRosetta Predictions (SigmaAldrich Product# NM_012110) murine RosettaPredictions (SigmaAldrich Product# NM_028850) PCBP1 MISSION ® esiRNAtargeting mouse Pcbp1 (esiRNA1) (SigmaAldrich Product# EMU011551) humanRosetta Predictions (SigmaAldrich Product# NM_006196) murine RosettaPredictions (SigmaAldrich Product# NM_011865) PBRM1 MISSION ® esiRNAhuman PBRM1 (esiRNA1) (SigmaAldrich Product# EHU075001) human RosettaPredictions (SigmaAldrich Product# NM_018165) human Rosetta Predictions(SigmaAldrich Product# NM_018313) human Rosetta Predictions(SigmaAldrich Product# NM_181042) WDR6 MISSION ® esiRNA human WDR6(esiRNA1) (SigmaAldrich Product# EHU065441) MISSION ® esiRNA targetingmouse Wdr6 (esiRNA1) (SigmaAldrich Product# EMU038981) human RosettaPredictions (SigmaAldrich Product# NM_018031) murine Rosetta Predictions(SigmaAldrich Product# NM_031392) E2F8 MISSION ® esiRNA human E2F8(esiRNA1) (SigmaAldrich Product# EHU025641) MISSION ® esiRNA targetingmouse E2f8 (SigmaAldrich Product# EMU206861) human Rosetta Predictions(SigmaAldrich Product# NM_024680) murine Rosetta Predictions(SigmaAldrich Product# NM_001013368) SERPINA3 MISSION ® esiRNA humanSERPINA3 (esiRNA1) (SigmaAldrich Product# EHU150301) human RosettaPredictions (SigmaAldrich Product# NM_001085) GNAS MISSION ® esiRNAhuman GNAS (esiRNA1) (SigmaAldrich Product# EHU117321) MISSION ® esiRNAtargeting mouse Gnas (esiRNA1) (SigmaAldrich Product# EMU074141) humanRosetta Predictions (SigmaAldrich Product# NM_000516) human RosettaPredictions (SigmaAldrich Product# NM_001077488) human RosettaPredictions (SigmaAldrich Product# NM_001077489) GNAS human RosettaPredictions (SigmaAldrich Product# NM_001077490) human RosettaPredictions (SigmaAldrich Product# NM_016592) ZC3H12A MISSION ® esiRNAtargeting human ZC3H12A (esiRNA1) (SigmaAldrich# EHU009491) MISSION ®esiRNA targeting mouse Zc3h12a (esiRNA1) (SigmaAldrich# EMU048551)Rosetta Predictions human (SigmaAldrich# NM_025079) Rosetta Predictionsmouse (SigmaAldrich# NM_153159) ZC3H12A Accell Mouse Zc3h12a (230738)siRNA - SMARTpool (Dharmacon# E- 052076-00-0005) MAP4K1 MISSION ® esiRNAtargeting human MAP4K1 (esiRNA1) (SigmaAldrich# EHU005191) MISSION ®esiRNA targeting mouse Map4kl (esiRNA1) (SigmaAldrich# EMU086771)Rosetta Predictions human (SigmaAldrich# NM_001042600) RosettaPredictions human (SigmaAldrich# NM_007181) Rosetta Predictions mouse(SigmaAldrich# NM_008279) NR4A3 MISSION ® esiRNA targeting human NR4A3(esiRNA1) (SigmaAldrich# EHU014951) Rosetta Predictions human(SigmaAldrich# NM_006981) Rosetta Predictions human (SigmaAldrich#NM_173198) Rosetta Predictions human (SigmaAldrich# NM_173199) RosettaPredictions human (SigmaAldrich# NM_173200)

TABLE 4B Exemplary shRNA constructs Target Gene shRNA construct SEMA7AMISSION ® shRNA murine Plasmid DNA (SigmaAldrich Product#SHCLND-NM_011352) MISSION ® shRNA human Plasmid DNA (SigmaAldrichProduct# SHCLND-NM_003612) RBM39 MISSION ® shRNA murine Plasmid DNA(SigmaAldrich Product# SHCLND-NM_133242) MISSION ® shRNA human PlasmidDNA (SigmaAldrich Product# SHCLND-NM_004902) BCL2L11 MISSION ® shRNAmurine Plasmid DNA (SigmaAldrich Product# SHCLND-NM_009754) MISSION ®shRNA human Plasmid DNA (SigmaAldrich Product# SHCLND-NM_138621) FLI1MISSION ® shRNA human Plasmid DNA (SigmaAldrich Product#SHCLND-NM_002017 MISSION ® shRNA murine Plasmid DNA (SigmaAldrichProduct# SHCLND-NM_008026) CALM2 MISSION ® shRNA murine Plasmid DNA(SigmaAldrich Product# SHCLND-NM_007589) MISSION ® shRNA human PlasmidDNA (SigmaAldrich Product# SHCLND-NM_001743) DHODH MISSION ® shRNAmurine Plasmid DNA (SigmaAldrich Product# SHCLND-NM_020046) MISSION ®shRNA human Plasmid DNA (SigmaAldrich Product# SHCLND-NM_001361) UMPSMISSION ® shRNA murine Plasmid DNA (SigmaAldrich Product#SHCLND-NM_009471) MISSION ® shRNA human Plasmid DNA (SigmaAldrichProduct# SHCLND-NM_000373) CHIC2 MISSION ® shRNA murine Plasmid DNA(SigmaAldrich Product# SHCLND-NM_028850) MISSION ® shRNA human PlasmidDNA (SigmaAldrich Product# SHCLND-NM_012110) PCBP1 MISSION ® shRNAmurine Plasmid DNA (SigmaAldrich Product# SHCLND-NM_011865) MISSION ®shRNA human Plasmid DNA (SigmaAldrich Product# SHCLND-NM_006196) PBRM1MISSION ® shRNA murine Plasmid DNA (SigmaAldrich Product#SHCLND-NM_001081251) PBRM1 MISSION ® shRNA human Plasmid DNA(SigmaAldrich Product# SHCLND-NM_018165) WDR6 MISSION ® shRNA murinePlasmid DNA (SigmaAldrich Product# SHCLND-NM_031392) MISSION ® shRNAhuman Plasmid DNA (SigmaAldrich Product# SHCLND-NM_018031) E2F8MISSION ® shRNA murine Plasmid DNA (SigmaAldrich Product#SHCLND-NM_001013368) MISSION ® shRNA human Plasmid DNA (SigmaAldrichProduct# SHCLND-NM_024680) SERPINA3 MISSION ® shRNA human Plasmid DNA(SigmaAldrich Product# SHCLND-NM_001085) GNAS MISSION ® shRNA murinePlasmid DNA (SigmaAldrich Product# SHCLND-NM_010309) MISSION ® shRNAhuman Plasmid DNA (SigmaAldrich Product# SHCLND-NM_000516) ZC3H12AMISSION ® shRNA Plasmid DNA human (SigmaAldrich# SHCLND-NM_025079)MISSION ® shRNA Plasmid DNA mouse (SigmaAldrich# SHCLND-NM_153159)MAP4K1 MISSION ® shRNA Plasmid DNA human (SigmaAldrich#SHCLND-NM_007181) MISSION ® shRNA Plasmid DNA mouse (SigmaAldrich#SHCLND-NM_008279) NR4A3 MISSION ® shRNA Plasmid DNA human (SigmaAldrich#SHCLND-NM_006981) MISSION ® shRNA Plasmid DNA mouse (SigmaAldrich#SHCLND-NM_015743)

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules (e.g., two or more siRNAs, two or more shRNAs,two or more RNA aptamers, or two or more morpholinos), wherein at leastone of the nucleic acid molecules binds to a target RNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto an RNA sequence encoded by a DNA sequence of a target gene selectedfrom IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR (e.g., a gene selected fromTable 2) and wherein at least one of the nucleic acid molecules binds toa target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to an RNA sequence encoded by a DNAsequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS,ZC3H12A, MAP4K1, and NR4A3 (e.g., a gene selected from Table 3).

In some embodiments, at least one of the two or more nucleic acidmolecules to a target RNA sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to an RNA sequence encodedby a DNA sequence defined by a set of genomic coordinates shown in Table5A or Table 5B and at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by a DNA sequence defined by a set of genomic coordinates shownin Table 6A-Table 6H. In some embodiments, at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical anRNA sequence encoded by one of SEQ ID NOs: 814-1566 and at least one ofthe two or more nucleic acid molecules binds to a target RNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical an RNA sequence encoded by one of SEQ ID NOs: 154-498 or SEQID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence of atarget gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR andwherein at least one of the nucleic acid molecules binds to a target RNAsequence encoded by a DNA sequence of a target gene selected fromBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, at least one of thetwo or more nucleic acid molecules to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by a DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B and at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by a DNA sequence defined by a set of genomiccoordinates shown in Table 6A or Table 6B. In some embodiments, at leastone of the two or more nucleic acid molecules binds to a target RNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to an RNA sequence encoded by one of SEQ ID NOs:814-1064 and at least one of the two or more nucleic acid moleculesbinds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to an RNA sequence encoded by oneof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence ofCBLB and wherein at least one of the nucleic acid molecules binds to atarget RNA sequence encoded by a DNA sequence of a target gene selectedfrom BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1,PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, at least oneof the two or more nucleic acid molecules binds to a target RNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to an RNA sequence encoded by one of SEQ ID NOs: 814-1064 andat least one of the two or more nucleic acid molecules binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to an RNA sequence encoded by one of SEQ ID NOs:499-524.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence of atarget gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR andwherein at least one of the nucleic acid molecules binds to a target RNAsequence encoded by a DNA sequence of ZC3H12A. In some embodiments, atleast one of the two or more nucleic acid molecules to a target RNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to an RNA sequence encoded by a DNA sequence definedby a set of genomic coordinates shown in Table 5A or Table 5B and atleast one of the two or more nucleic acid molecules binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to an RNA sequence encoded by a DNA sequencedefined by a set of genomic coordinates shown in Table 6C or Table 6D.In some embodiments, at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 1065-1509 and at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813 In some embodiments, the gene-regulating system comprises two ormore nucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence ofthe CBLB gene and wherein at least one of the nucleic acid moleculesbinds to a target RNA sequence encoded by a DNA sequence of the ZC3H12Agene. In some embodiments, at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 1065-1509 and at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 499-524. In someembodiments, at least one of the two or more nucleic acid moleculesbinds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to an RNA sequence encoded by oneof SEQ ID NOs: 1065-1264 and at least one of the two or more nucleicacid molecules binds to a target RNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence of atarget gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR andwherein at least one of the nucleic acid molecules binds to a target RNAsequence encoded by a DNA sequence of MAP4K1. In some embodiments, atleast one of the two or more nucleic acid molecules to a target RNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to an RNA sequence encoded by a DNA sequence definedby a set of genomic coordinates shown in Table 5A or Table 5B and atleast one of the two or more nucleic acid molecules binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to an RNA sequence encoded by a DNA sequencedefined by a set of genomic coordinates shown in Table 6E or Table 6F.In some embodiments, at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 510-1538 and at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence ofthe CBLB gene and wherein at least one of the nucleic acid moleculesbinds to a target RNA sequence encoded by a DNA sequence of MAP4K1. Insome embodiments, at least one of the two or more nucleic acid moleculesbinds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to an RNA sequence encoded by oneof SEQ ID NOs: 510-1538 and at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence of atarget gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3,NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB,PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR andwherein at least one of the nucleic acid molecules binds to a target RNAsequence encoded by a DNA sequence of NR4A3. In some embodiments, atleast one of the two or more nucleic acid molecules to a target RNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to an RNA sequence encoded by a DNA sequence definedby a set of genomic coordinates shown in Table 5A or Table 5B and atleast one of the two or more nucleic acid molecules binds to a targetRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to an RNA sequence encoded by a DNA sequencedefined by a set of genomic coordinates shown in Table 6G or Table 6H.In some embodiments, at least one of the two or more nucleic acidmolecules binds to a target RNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 1539-1566 and at least one of the two ormore nucleic acid molecules binds to a target RNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toan RNA sequence encoded by one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813.

In some embodiments, the gene-regulating system comprises two or morenucleic acid molecules, wherein at least one of the nucleic acidmolecules binds to a target RNA sequence encoded by a DNA sequence ofthe CBLB gene and wherein at least one of the nucleic acid moleculesbinds to a target RNA sequence encoded by a DNA sequence of NR4A3. Insome embodiments, at least one of the two or more nucleic acid moleculesbinds to a target RNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to an RNA sequence encoded by oneof SEQ ID NOs: 1539-1566 and at least one of the two or more nucleicacid molecules binds to a target RNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to an RNA sequenceencoded by one of SEQ ID NOs: 499-524.

B. Protein-Based Gene-Regulating Systems

In some embodiments, a protein-based gene-regulating system is a systemcomprising one or more proteins capable of regulating the expression ofan endogenous target gene in a sequence specific manner without therequirement for a nucleic acid guide molecule. In some embodiments, theprotein-based gene-regulating system comprises a protein comprising oneor more zinc-finger binding domains and an enzymatic domain. In someembodiments, the protein-based gene-regulating system comprises aprotein comprising a Transcription activator-like effector nuclease(TALEN) domain and an enzymatic domain. Such embodiments are referred toherein as “TALENs”.

1. Zinc Finger Systems

Zinc finger-based systems comprise a fusion protein comprising twoprotein domains: a zinc finger DNA binding domain and an enzymaticdomain. A “zinc finger DNA binding domain”, “zinc finger protein”, or“ZFP” is a protein, or a domain within a larger protein, that binds DNAin a sequence-specific manner through one or more zinc fingers, whichare regions of amino acid sequence within the binding domain whosestructure is stabilized through coordination of a zinc ion. The zincfinger domain, by binding to a target DNA sequence, directs the activityof the enzymatic domain to the vicinity of the sequence and, hence,induces modification of the endogenous target gene in the vicinity ofthe target sequence. A zinc finger domain can be engineered to bind tovirtually any desired sequence. Accordingly, after identifying a targetgenetic locus containing a target DNA sequence at which cleavage orrecombination is desired (e.g., a target locus in a target genereferenced in Tables 2 or 3), one or more zinc finger binding domainscan be engineered to bind to one or more target DNA sequences in thetarget genetic locus. Expression of a fusion protein comprising a zincfinger binding domain and an enzymatic domain in a cell, effectsmodification in the target genetic locus.

In some embodiments, a zinc finger binding domain comprises one or morezinc fingers. Miller et al. (1985) EMBO J. 4:1609-1614; Rhodes (1993)Scientific American February: 56-65; U.S. Pat. No. 6,453,242. Typically,a single zinc finger domain is about 30 amino acids in length. Anindividual zinc finger binds to a three-nucleotide (i.e., triplet)sequence (or a four-nucleotide sequence which can overlap, by onenucleotide, with the four-nucleotide binding site of an adjacent zincfinger). Therefore the length of a sequence to which a zinc fingerbinding domain is engineered to bind (e.g., a target sequence) willdetermine the number of zinc fingers in an engineered zinc fingerbinding domain. For example, for ZFPs in which the finger motifs do notbind to overlapping subsites, a six-nucleotide target sequence is boundby a two-finger binding domain; a nine-nucleotide target sequence isbound by a three-finger binding domain, etc. Binding sites forindividual zinc fingers (i.e., subsites) in a target site need not becontiguous, but can be separated by one or several nucleotides,depending on the length and nature of the amino acids sequences betweenthe zinc fingers (i.e., the inter-finger linkers) in a multi-fingerbinding domain. In some embodiments, the DNA-binding domains ofindividual ZFNs comprise between three and six individual zinc fingerrepeats and can each recognize between 9 and 18 basepairs.

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection.

Selection of a target DNA sequence for binding by a zinc finger domaincan be accomplished, for example, according to the methods disclosed inU.S. Pat. No. 6,453,242. It will be clear to those skilled in the artthat simple visual inspection of a nucleotide sequence can also be usedfor selection of a target DNA sequence. Accordingly, any means fortarget DNA sequence selection can be used in the methods describedherein. A target site generally has a length of at least 9 nucleotidesand, accordingly, is bound by a zinc finger binding domain comprising atleast three zinc fingers. However binding of, for example, a 4-fingerbinding domain to a 12-nucleotide target site, a 5-finger binding domainto a 15-nucleotide target site or a 6-finger binding domain to an18-nucleotide target site, is also possible. As will be apparent,binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) tolonger target sites is also possible.

In some embodiments, the zinc finger binding domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of a target gene selectedfrom IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR (e.g., a gene selected fromTable 2). In some embodiments, the zinc finger binding domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in Table 5A or Table 5B. In someembodiments, the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the zinc finger binding domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of CBLB. In someembodiments, the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 499-524. In some embodiments,the zinc finger binding domains bind to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toa target DNA sequence of BCOR. In some embodiments, the zinc fingerbinding domains bind to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQ IDNOs: 708-772 or SEQ ID NOs: 708-764. In some embodiments, the zincfinger binding domains bind to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of TNFAIP3. In some embodiments, the zinc fingerbinding domains bind to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQ IDNOs: 348-396 or SEQ ID NOs: 348-386.

In some embodiments, the zinc finger binding domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of a target gene selectedBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., a geneselected from Table 3). In some embodiments, the zinc finger bindingdomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in one of Table6A-Table 6H. In some embodiments, the zinc finger binding domains bindto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to one of SEQ ID NOs: 814-1566.

In some embodiments, the zinc finger binding domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of a target gene selectedBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, the zinc fingerbinding domains bind to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6A orTable 6B. In some embodiments, the zinc finger binding domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 814-1064. In someembodiments, the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of ZC3H12A. In someembodiments, the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6C or Table 6D. In some embodiments, the zincfinger binding domains bind to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1509. In some embodiments, the zinc finger bindingdomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1065-1264.

In some embodiments, the zinc finger binding domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of the MAP4K1 gene. Insome embodiments, the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6E or Table 6F. In some embodiments, the zincfinger binding domains bind to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 510-1538. In some embodiments, the zinc finger bindingdomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of the NR4A3 gene. In some embodiments, the zinc finger bindingdomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6G orTable 6H. In some embodiments, the zinc finger binding domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1539-1566.

In some embodiments, the zinc finger system is selected from those knownin the art, such as those available from commercial suppliers such asSigma Aldrich. For example, in some embodiments, the zinc finger systemis selected from those known in the art, such as those described inTable 7 below.

TABLE 7 Exemplary Zinc Finger Systems Target Gene Zinc Finger SystemSEMA7A CompoZr ® Knockout ZFN plasmid human SEMA7A NM_003612(SigmaAldrich Product# CKOZFND19082) CompoZr ® Knockout ZFN plasmidmurine Sema7a NM_011352.2 (SigmaAldrich Product# CKOZFND 19082) RBM39CompoZr ® Knockout ZFN plasmid Human RBM39 (NM_004902) (SigmaAldrichProduct# CKOZFND 18044) CompoZr ® Knockout ZFN plasmid Mouse Rbm39(NM_133242.2) (SigmaAldrich Product # CKOZFND39983) BCL2L11 CompoZr ®Knockout ZFN plasmid Human BCL2L11 (NM_006538) (SigmaAldrich Product #CKOZFND3909) CompoZr ® Knockout ZFN plasmid Mouse Bcl2l11 (NM_207680.2)(SigmaAldrich Product # CKOZFND27562) FLI1 CompoZr ® Knockout ZFN KitHuman FLI1 (NM_002017) (SigmaAldrich Product# CKOZFN8731) FLI1 CompoZr ®Knockout ZFN plasmid Mouse Fli1 (NM_008026.4) (SigmaAldrich Product#CKOZFND31430) CALM2 CompoZr ® Knockout ZFN Kit Human CALM2 (NM_001743)(SigmaAldrich Product# CKOZFN5301) CompoZr ® Knockout ZFN plasmid MouseCalm2 (NM_007589.5) (SigmaAldrich Product# CKOZFND27915) DHODH CompoZr ®Knockout ZFN plasmid Human DHODH (NM_001361) (SigmaAldrich Product #CKOZFND 1982) CompoZr ® Knockout ZFN plasmid Mouse Dhodh (NM_020046.3)(SigmaAldrich Product # CKOZFND29960) UMPS CompoZr ® Knockout ZFNplasmid Human UMPS (NM_000373) (SigmaAldrich Product# CKOZFND1693)CompoZr ® Knockout ZFN plasmid Mouse Umps (NM_009471.2) (SigmaAldrichProduct# CKOZFND43931) CHIC2 CompoZr ® Knockout ZFN Kit Human CHIC2(NM_012110) (SigmaAldrich Product # CKOZFN6059) CompoZr ® Knockout ZFNplasmid Mouse Chic2 (NM_028850.4) (SigmaAldrich Product# CKOZFND28691)PCBP1 CompoZr ® Knockout ZFN plasmid Human PCBP1 (NM_006196)(SigmaAldrich Product# CKOZFND16392) CompoZr ® Knockout ZFN plasmidMouse Pcbp1 (NM_011865.3) (SigmaAldrich Product# CKOZFND38313) PBRM1CompoZr ® Knockout ZFN plasmid Human PBRM1 (NM_018165) (SigmaAldrichProduct # CKOZFND2434) CompoZr ® Knockout ZFN plasmid Mouse Pbrm1(NM_001081251.1) (SigmaAldrich Product # CKOZFND38304) WDR6 CompoZr ®Knockout ZFN plasmid Human WDR6 (NM_018031) (SigmaAldrich Product#CKOZFND22841) CompoZr ® Knockout ZFN plasmid Mouse Wdr6 (NM_031392.2)(SigmaAldrich Product # CKOZFND44594) E2F8 CompoZr ® Knockout ZFNplasmid Human E2F8 (NM_024680) (SigmaAldrich Product# CKOZFND7610)CompoZr ® Knockout ZFN plasmid Mouse E2f8 (NM_001013368.5) (SigmaAldrichProduct # CKOZFND30371) SERPINA3 CompoZr ® Knockout ZFN plasmid HumanSERPINA3 (NM_001085) (SigmaAldrich Product # CKOZFND1900) GNAS CompoZr ®Knockout ZFN plasmid Human GNAS (NM_000516) (SigmaAldrich Product#CKOZFND1354) CompoZr ® Knockout ZFN plasmid Mouse Gnas (NM_001077510.2)(SigmaAldrich Product # CKOZFND32583) ZC3H12A CompoZr ® Knockout ZFNKit, ZFN plasmid Human ZC3H12A (NM_025079) (SigmaAldrich# CKOZFND23094)CompoZr ® Knockout ZFN Kit, ZFN plasmid mouse Zc3h12a (NM_153159.2)(SigmaAldrich# CKOZFND44851) MAP4K1 CompoZr ® Knockout ZFN plasmid HumanMAP4K1 (NM_001042600) (SigmaAldrich# CKOZFND12999) CompoZr ® KnockoutZFN plasmid Mouse Map4k1 (NM_008279.2) (SigmaAldrich# CKOZFND35246)NR4A3 CompoZr ® Knockout ZFN plasmid Human NR4A3 (NM_006981)(SigmaAldrich# CKOZFND2362) CompoZr ® Knockout ZFN plasmid Mouse Nr4a3(NM_015743.2) (SigmaAldrich# CKOZFND36667)

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or 100% identical to a target DNA sequence of a target geneselected from IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2,TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1,HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR and wherein at leastone of the zinc finger binding domains binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to a target DNA sequence of a target gene selected fromBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3. In someembodiments, at least one of the zinc finger binding domains binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in Table 5A or Table 5B and at leastone of the zinc finger binding domains binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to a target DNA sequence defined by a set of genomiccoordinates shown in one of Tables 6A-Table 6H. In some embodiments, atleast one of the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813and at least one of the zinc finger binding domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 814-1566.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence a target gene selected from IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and at least one of the zinc finger binding domains bindsto a target DNA sequence of a target gene selected from BCL2L11, FLI1,CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8,SERPINA3, and GNAS. In some embodiments, at least one of the two or morezinc finger binding domains binds to a target DNA sequence that is atleast 90% 95%, 96%, 97%, 98%, or 99% identical, or 100% identical to atarget DNA sequence defined by a set of genomic coordinates shown inTable 5A or Table 5B and at least one of the two or more zinc fingerbinding domains binds to a target DNA sequence that is at least 90% 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6A orTable 6B. In some embodiments, at least one of the two or more zincfinger binding domains binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 814-1064 and at least one of the two or more zinc fingerbinding domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:814-1064 and at least one of the two or more zinc finger binding domainsbinds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to one of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence a target gene selected from IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and at least one of the zinc finger binding domains bindsto a target DNA sequence of ZC3H12A. In some embodiments, at least oneof the two or more zinc finger binding domains binds to a target DNAsequence that is at least 90% 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B and at least one of the two ormore zinc finger binding domains binds to a target DNA sequence that isat least 90% 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence defined by a set of genomic coordinates shownin Table 6C or Table 6D. In some embodiments, at least one of the two ormore zinc finger binding domains binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto one of SEQ ID NOs: 1065-1509 and at least one of the two or more zincfinger binding domains binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence the CBLB gene and at least one of the zinc fingerbinding domains binds to a target DNA sequence of the ZC3H12A gene. Insome embodiments, at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1065-1509 and at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524. In some embodiments, at least one of the two or more zincfinger binding domains binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1264 and at least one of the two or more zinc fingerbinding domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence a target gene selected from IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and at least one of the zinc finger binding domains bindsto a target DNA sequence of the MAP4K1 gene. In some embodiments, atleast one of the two or more zinc finger binding domains binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in Table 5A or Table 5B and at leastone of the two or more zinc finger binding domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6D or Table 6E. In some embodiments, at leastone of the two or more zinc finger binding domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 510-1538 and at least one of thetwo or more zinc finger binding domains binds to a target DNA sequencethat is at least at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence the CBLB gene selected and at least one of the zincfinger binding domains binds to a target DNA sequence of the MAP4K1gene. In some embodiments, at least one of the two or more zinc fingerbinding domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 510-1538 and at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence a target gene selected from IKZF1, IKZF3, GATA3,BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and at least one of the zinc finger binding domains bindsto a target DNA sequence of the NR4A3 gene. In some embodiments, atleast one of the two or more zinc finger binding domains binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in Table 5A or Table 5B and at leastone of the two or more zinc finger binding domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6F or Table 6G. In some embodiments, at leastone of the two or more zinc finger binding domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 1539-1566 and at least one ofthe two or more zinc finger binding domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreZFP-fusion proteins each comprising a zinc finger binding domain,wherein at least one of the zinc finger binding domains binds to atarget DNA sequence the CBLB gene selected and at least one of the zincfinger binding domains binds to a target DNA sequence of the NR4A3 gene.In some embodiments, at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1539-1566 and at least one of the two or more zinc finger bindingdomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524.

The enzymatic domain portion of the zinc finger fusion proteins can beobtained from any endo- or exonuclease. Exemplary endonucleases fromwhich an enzymatic domain can be derived include, but are not limitedto, restriction endonucleases and homing endonucleases. See, forexample, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; andBelfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additionalenzymes which cleave DNA are known (e.g., 51 Nuclease; mung beannuclease; pancreatic DNaseI; micrococcal nuclease; yeast HOendonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring HarborLaboratory Press, 1993). One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains.

Exemplary restriction endonucleases (restriction enzymes) suitable foruse as an enzymatic domain of the ZFPs described herein are present inmany species and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise theenzymatic domain from at least one Type IIS restriction enzyme and oneor more zinc finger binding domains.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Thus, for targeted double-stranded DNA cleavage usingzinc finger-FokI fusions, two fusion proteins, each comprising a FokIenzymatic domain, can be used to reconstitute a catalytically activecleavage domain. Alternatively, a single polypeptide molecule containinga zinc finger binding domain and two FokI enzymatic domains can also beused. Exemplary ZFPs comprising FokI enzymatic domains are described inU.S. Pat. No. 9,782,437.

2. TALEN Systems

TALEN-based systems comprise a protein comprising a TAL effector DNAbinding domain and an enzymatic domain. They are made by fusing a TALeffector DNA-binding domain to a DNA cleavage domain (a nuclease whichcuts DNA strands). The FokI restriction enzyme described above is anexemplary enzymatic domain suitable for use in TALEN-basedgene-regulating systems.

TAL effectors are proteins that are secreted by Xanthomonas bacteria viatheir type III secretion system when they infect plants. The DNA bindingdomain contains a repeated, highly conserved, 33-34 amino acid sequencewith divergent 12th and 13th amino acids. These two positions, referredto as the Repeat Variable Diresidue (RVD), are highly variable andstrongly correlated with specific nucleotide recognition. Therefore, theTAL effector domains can be engineered to bind specific target DNAsequences by selecting a combination of repeat segments containing theappropriate RVDs. The nucleic acid specificity for RVD combinations isas follows: HD targets cytosine, NI targets adenenine, NG targetsthymine, and NN targets guanine (though, in some embodiments, NN canalso bind adenenine with lower specificity).

In some embodiments, the TAL effector domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of a target gene selectedfrom IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAGS, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR (e.g., a gene selected fromTable 2). In some embodiments, the TAL effector domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence defined by a set ofgenomic coordinates shown in Table 5A or Table 5B. In some embodiments,the TAL effector domains bind to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments, theTAL effector domains bind to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a targetDNA sequence of the CBLB gene. In some embodiments, the TAL effectordomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524. In some embodiments, the TAL effector domains bind to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to a target DNA sequence of the BCOR gene, and bindto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to one of SEQ ID NOs: 708-772 or SEQID NOs: 708-764. In some embodiments, the TAL effector domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence of the TNFAIP3,bind to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to one of SEQ ID NOs: 348-396 orSEQ ID NOs: 348-386.

In some embodiments, the TAL effector domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of a target gene selectedfrom BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1,PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., agene selected from Table 3). In some embodiments, the TAL effectordomains bind to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in one of Tables6A-Table 6H. In some embodiments, the TAL effector domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 814-1566.

In some embodiments, the TAL effector domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of a target gene selectedfrom BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1,PBRM1, WDR6, E2F8, SERPINA3, and GNAS. In some embodiments, the TALeffector domains bind to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a targetDNA sequence defined by a set of genomic coordinates shown in Table 6Aor Table 6B. In some embodiments, the TAL effector domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 814-1064. In someembodiments, the TAL effector domains bind to a target DNA sequence thatis at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to a target DNA sequence of ZC3H12A. In some embodiments, theTAL effector domains bind to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a targetDNA sequence defined by a set of genomic coordinates shown in Table 6Cor Table 6D. In some embodiments, the TAL effector domains bind to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1065-1509. In someembodiments, the TAL effector domains bind to a target DNA sequence thatis at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 1065-1264.

In some embodiments, the TAL effector domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of the MAP4K1 gene. In someembodiments, the TAL effector domains bind to a target DNA sequence thatis at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6E or Table 6F. In some embodiments, the TALeffector domains bind to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 510-1538. In some embodiments, the TAL effector domains bind toa target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence of the NR4A3gene. In some embodiments, the TAL effector domains bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6G or Table 6H. In some embodiments, the TALeffector domains bind to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 1539-1566.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of a target gene selectedfrom IKZF1, IKZF3, GATA3, BCL3, TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1,TGFBR2, TANK, FOXP3, RC3H1, TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2,LAG3, TIGIT, CTLA4, PTPN6, PDCD1, or BCOR and at least one of the TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a targetDNA sequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS,ZC3H12A, MAP4K1, and NR4A3. In some embodiments, at least one of the TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to a targetDNA sequence defined by a set of genomic coordinates shown in Table 5Aor Table 5B and at least one of the TAL effector domains binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in one of Tables 6A-Table 6H. In someembodiments, at least one of the TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813 and at least one of the TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 814-1566.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence a target gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, orBCOR and at least one of the TAL effector domains binds to a target DNAsequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, andGNAS. In some embodiments, at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 5A orTable 5B and at least one of the two or more TAL effector domains bindsto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to a target DNA sequence defined bya set of genomic coordinates shown in Table 6A or Table 6B. In someembodiments, at least one of the two or more TAL effector domains bindsto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to one of SEQ ID NOs: 814-1064 andat least one of the two or more TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs:499-813.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence of CBLB and at least one of the TAL effector domains binds to atarget DNA sequence of a target gene selected from BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,and GNAS. In some embodiments, at least one of the two or more TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 814-1064 and at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence a target gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, orBCOR and at least one of the TAL effector domains binds to a target DNAsequence of ZC3H12A. In some embodiments, at least one of the two ormore TAL effector domains binds to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toa target DNA sequence defined by a set of genomic coordinates shown inTable 5A or Table 5B and at least one of the two or more TAL effectordomains binds binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6C orTable 6D. In some embodiments, at least one of the two or more TALeffector domains binds binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1509 and at least one of the two or more TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence the CBLB gene and at least one of the TAL effector domainsbinds to a target DNA sequence of the ZC3H12A gene. In some embodiments,at least one of the two or more TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 1065-1509 and at least one ofthe two or more TAL effector domains binds to a target DNA sequence thatis at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 499-524. In some embodiments, at leastone of the two or more TAL effector domains binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 1065-1264 and at least one ofthe two or more TAL effector domains binds to a target DNA sequence thatis at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence a target gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, orBCOR and at least one of the TAL effector domains binds to a target DNAsequence of the MAP4K1 gene. In some embodiments, at least one of thetwo or more TAL effector domains binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence defined by a set of genomic coordinates shownin Table 5A or Table 5B and at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6D orTable 6E. In some embodiments, at least one of the two or more TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 510-1538 and at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence of the CBLB gene selected and at least one of the TAL effectordomains binds to a target DNA sequence of the MAP4K1 gene. In someembodiments, at least one of the two or more TAL effector domains bindsto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to one of SEQ ID NOs: 510-1538 andat least one of the two or more TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 499-524.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence a target gene selected from IKZF1, IKZF3, GATA3, BCL3, TNIP1,TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1, TRAF6,IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6, PDCD1, orBCOR and at least one of the TAL effector domains binds to a target DNAsequence of the NR4A3 gene. In some embodiments, at least one of the twoor more TAL effector domains binds to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toa target DNA sequence defined by a set of genomic coordinates shown inTable 5A or Table 5B and at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6F orTable 6G. In some embodiments, at least one of the two or more TALeffector domains binds to a target DNA sequence that is at least 90%,95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQID NOs: 1539-1566 and at least one of the two or more TAL effectordomains binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:154-498 or SEQ ID NOs: 499-813.

In some embodiments, the gene-regulating system comprises two or moreTAL effector-fusion proteins each comprising a TAL effector domain,wherein at least one of the TAL effector domains binds to a target DNAsequence of the CBLB gene selected and at least one of the TAL effectordomains binds to a target DNA sequence of the NR4A3 gene. In someembodiments, at least one of the two or more TAL effector domains bindsto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to one of SEQ ID NOs: 1539-1566 andat least one of the two or more TAL effector domains binds to a targetDNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical,or is 100% identical to one of SEQ ID NOs: 499-524.

Methods and compositions for assembling the TAL-effector repeats areknown in the art. See e.g., Cermak et al, Nucleic Acids Research, 39:12,2011, e82. Plasmids for constructions of the TAL-effector repeats arecommercially available from Addgene.

C. Combination Nucleic Acid/Protein-Based Gene-Regulating Systems

Combination gene-regulating systems comprise a site-directed modifyingpolypeptide and a nucleic acid guide molecule. Herein, a “site-directedmodifying polypeptide” refers to a polypeptide that binds to a nucleicacid guide molecule, is targeted to a target nucleic acid sequence, (forexample, an endogenous target DNA or RNA sequence) by the nucleic acidguide molecule to which it is bound, and modifies the target nucleicacid sequence (e.g., by cleavage, mutation, or methylation of the targetnucleic acid sequence).

A site-directed modifying polypeptide comprises two portions, a portionthat binds the nucleic acid guide and an activity portion. In someembodiments, a site-directed modifying polypeptide comprises an activityportion that exhibits site-directed enzymatic activity (e.g., DNAmethylation, DNA or RNA cleavage, histone acetylation, histonemethylation, etc.), wherein the site of enzymatic activity is determinedby the guide nucleic acid. In some cases, a site-directed modifyingpolypeptide comprises an activity portion that has enzymatic activitythat modifies the endogenous target nucleic acid sequence (e.g.,nuclease activity, methyltransferase activity, demethylase activity, DNArepair activity, DNA damage activity, deamination activity, dismutaseactivity, alkylation activity, depurination activity, oxidationactivity, pyrimidine dimer forming activity, integrase activity,transposase activity, recombinase activity, polymerase activity, ligaseactivity, helicase activity, photolyase activity or glycosylaseactivity). In other cases, a site-directed modifying polypeptidecomprises an activity portion that has enzymatic activity that modifiesa polypeptide (e.g., a histone) associated with the endogenous targetnucleic acid sequence (e.g., methyltransferase activity, demethylaseactivity, acetyltransferase activity, deacetylase activity, kinaseactivity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity,SUMOylating activity, deSUMOylating activity, ribosylation activity,deribosylation activity, myristoylation activity or demyristoylationactivity). In some embodiments, a site-directed modifying polypeptidecomprises an activity portion that modulates transcription of a targetDNA sequence (e.g., to increase or decrease transcription). In someembodiments, a site-directed modifying polypeptide comprises an activityportion that modulates expression or translation of a target RNAsequence (e.g., to increase or decrease transcription).

The nucleic acid guide comprises two portions: a first portion that iscomplementary to, and capable of binding with, an endogenous targetnucleic sequence (referred to herein as a “nucleic acid-bindingsegment”), and a second portion that is capable of interacting with thesite-directed modifying polypeptide (referred to herein as a“protein-binding segment”). In some embodiments, the nucleicacid-binding segment and protein-binding segment of a nucleic acid guideare comprised within a single polynucleotide molecule. In someembodiments, the nucleic acid-binding segment and protein-bindingsegment of a nucleic acid guide are each comprised within separatepolynucleotide molecules, such that the nucleic acid guide comprises twopolynucleotide molecules that associate with each other to form thefunctional guide.

The nucleic acid guide mediates the target specificity of the combinedprotein/nucleic acid gene-regulating systems by specifically hybridizingwith a target nucleic acid sequence. In some embodiments, the targetnucleic acid sequence is an RNA sequence, such as an RNA sequencecomprised within an mRNA transcript of a target gene. In someembodiments, the target nucleic acid sequence is a DNA sequencecomprised within the DNA sequence of a target gene. Reference herein toa target gene encompasses the full-length DNA sequence for thatparticular gene which comprises a plurality of target genetic loci(i.e., portions of a particular target gene sequence (e.g., an exon oran intron)). Within each target genetic loci are shorter stretches ofDNA sequences referred to herein as “target DNA sequences” that can bemodified by the gene-regulating systems described herein. Further, eachtarget genetic loci comprises a “target modification site,” which refersto the precise location of the modification induced by thegene-regulating system (e.g., the location of an insertion, a deletion,or mutation, the location of a DNA break, or the location of anepigenetic modification).

The gene-regulating systems described herein may comprise a singlenucleic acid guide, or may comprise a plurality of nucleic acid guides(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid guides).

In some embodiments, the combined protein/nucleic acid gene-regulatingsystems comprise site-directed modifying polypeptides derived fromArgonaute (Ago) proteins (e.g., T. thermophiles Ago or TtAgo). In suchembodiments, the site-directed modifying polypeptide is a T.thermophiles Ago DNA endonuclease and the nucleic acid guide is a guideDNA (gDNA) (See, Swarts et al., Nature 507 (2014), 258-261). In someembodiments, the present disclosure provides a polynucleotide encoding agDNA. In some embodiments, a gDNA-encoding nucleic acid is comprised inan expression vector, e.g., a recombinant expression vector. In someembodiments, the present disclosure provides a polynucleotide encoding aTtAgo site-directed modifying polypeptide or variant thereof. In someembodiments, the polynucleotide encoding a TtAgo site-directed modifyingpolypeptide is comprised in an expression vector, e.g., a recombinantexpression vector.

In some embodiments, the gene editing systems described herein areCRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR Associated) nuclease systems. In some embodiments, theCRISPR/Cas system is a Class 2 system. Class 2 CRISPR/Cas systems aredivided into three types: Type II, Type V, and Type VI systems. In someembodiments, the CRISPR/Cas system is a Class 2 Type II system,utilizing the Cas9 protein. In such embodiments, the site-directedmodifying polypeptide is a Cas9 DNA endonuclease (or variant thereof)and the nucleic acid guide molecule is a guide RNA (gRNA). In someembodiments, the CRISPR/Cas system is a Class 2 Type V system, utilizingthe Cas12 proteins (e.g., Cas12a (also known as Cpf1), Cas12b (alsoknown as C2c1), Cas12c (also known as C2c3), Cas12d (also known asCasY), and Cas12e (also known as CasX)). In such embodiments, thesite-directed modifying polypeptide is a Cas12 DNA endonuclease (orvariant thereof) and the nucleic acid guide molecule is a gRNA. In someembodiments, the CRISPR/Cas system is a Class 2 and Type VI system,utilizing the Cas13 proteins (e.g., Cas13a (also known as C2c2), Cas13b,and Cas13c). (See, Pyzocha et al., ACS Chemical Biology, 13(2),347-356). In such embodiments, the site-directed modifying polypeptideis a Cas13 RNA riboendonuclease and the nucleic acid guide molecule is agRNA.

A Cas polypeptide refers to a polypeptide that can interact with a gRNAmolecule and, in concert with the gRNA molecule, home or localize to atarget DNA or target RNA sequence. Cas polypeptides include naturallyoccurring Cas proteins and engineered, altered, or otherwise modifiedCas proteins that differ by one or more amino acid residues from anaturally-occurring Cas sequence.

A guide RNA (gRNA) comprises two segments, a DNA-binding segment and aprotein-binding segment. In some embodiments, the protein-bindingsegment of a gRNA is comprised in one RNA molecule and the DNA-bindingsegment is comprised in another separate RNA molecule. Such embodimentsare referred to herein as “double-molecule gRNAs” or “two-molecule gRNA”or “dual gRNAs.” In some embodiments, the gRNA is a single RNA moleculeand is referred to herein as a “single-guide RNA” or an “sgRNA.” Theterm “guide RNA” or “gRNA” is inclusive, referring both to two-moleculeguide RNAs and sgRNAs.

The protein-binding segment of a gRNA comprises, in part, twocomplementary stretches of nucleotides that hybridize to one another toform a double stranded RNA duplex (dsRNA duplex), which facilitatesbinding to the Cas protein. The nucleic acid-binding segment (or“nucleic acid-binding sequence”) of a gRNA comprises a nucleotidesequence that is complementary to and capable of binding to a specifictarget nucleic acid sequence. The protein-binding segment of the gRNAinteracts with a Cas polypeptide and the interaction of the gRNAmolecule and site-directed modifying polypeptide results in Cas bindingto the endogenous nucleic acid sequence and produces one or moremodifications within or around the target nucleic acid sequence. Theprecise location of the target modification site is determined by both(i) base-pairing complementarity between the gRNA and the target nucleicacid sequence; and (ii) the location of a short motif, referred to asthe protospacer adjacent motif (PAM), in the target DNA sequence(referred to as a protospacer flanking sequence (PFS) in target RNAsequences). The PAM/PFS sequence is required for Cas binding to thetarget nucleic acid sequence. A variety of PAM/PFS sequences are knownin the art and are suitable for use with a particular Cas endonuclease(e.g., a Cas9 endonuclease)(See e.g., Nat Methods. 2013 November;10(11): 1116-1121 and Sci Rep. 2014; 4: 5405). In some embodiments, thePAM sequence is located within 50 base pairs of the target modificationsite in a target DNA sequence. In some embodiments, the PAM sequence islocated within 10 base pairs of the target modification site in a targetDNA sequence. The DNA sequences that can be targeted by this method arelimited only by the relative distance of the PAM sequence to the targetmodification site and the presence of a unique 20 base pair sequence tomediate sequence-specific, gRNA-mediated Cas binding. In someembodiments, the PFS sequence is located at the 3′ end of the target RNAsequence. In some embodiments, the target modification site is locatedat the 5′ terminus of the target locus. In some embodiments, the targetmodification site is located at the 3′ end of the target locus. In someembodiments, the target modification site is located within an intron oran exon of the target locus.

In some embodiments, the present disclosure provides a polynucleotideencoding a gRNA. In some embodiments, a gRNA-encoding nucleic acid iscomprised in an expression vector, e.g., a recombinant expressionvector. In some embodiments, the present disclosure provides apolynucleotide encoding a site-directed modifying polypeptide. In someembodiments, the polynucleotide encoding a site-directed modifyingpolypeptide is comprised in an expression vector, e.g., a recombinantexpression vector.

1. Cas Proteins

In some embodiments, the site-directed modifying polypeptide is a Casprotein. Cas molecules of a variety of species can be used in themethods and compositions described herein, including Cas moleculesderived from S. pyogenes, S. aureus, N. meningitidis, S. thermophiles,Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillussuccinogenes, Actinobacillus suis, Actinomyces sp.,Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus,Bacillus smithii, Bacillus thuringiensis, Bacteroides sp.,Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterospoxus,Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatuspuniceispirillum, Clostridium cellulolyticum, Clostridium perfringens,Corynebacterium accolens, Corynebacterium diphtheria, Corynebacteriummatruchotii, Dinoroseobacter shibae, Eubacterium dolichum,Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilusparainjluenzae, Haemophilus sputomm, Helicobacter canadensis,Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus,Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeriamonocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinustrichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseriacinerea, Neisseria flavescens, Neisseria lactamica, Neisseriameningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp.,Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp.,Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcuslugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis,Treponema sp., or Verminephrobacter eiseniae.

In some embodiments, the Cas protein is a naturally-occurring Casprotein. In some embodiments, the Cas endonuclease is selected from thegroup consisting of C2C1, C2C3, Cpf1 (also referred to as Cas12a),Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1,Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known asCsn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5,Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1,Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1,Csf2, Csf3, and Csf4.

In some embodiments, the Cas protein is an endoribonuclease such as aCas13 protein. In some embodiments, the Cas13 protein is a Cas13a(Abudayyeh et al., Nature 550 (2017), 280-284), Cas13b (Cox et al.,Science (2017) 358:6336, 1019-1027), Cas13c (Cox et al., Science (2017)358:6336, 1019-1027), or Cas13d (Zhang et al., Cell 175 (2018), 212-223)protein.

In some embodiments, the Cas protein is a wild-type or naturallyoccurring Cas9 protein or a Cas9 ortholog. Wild-type Cas9 is amulti-domain enzyme that uses an HNH nuclease domain to cleave thetarget strand of DNA and a RuvC-like domain to cleave the non-targetstrand. Binding of WT Cas9 to DNA based on gRNA specificity results indouble-stranded DNA breaks that can be repaired by non-homologous endjoining (NHEJ) or homology-directed repair (HDR). Exemplary naturallyoccurring Cas9 molecules are described in Chylinski et al., RNA Biology2013 10:5, 727-737 and additional Cas9 orthologs are described inInternational PCT Publication No. WO 2015/071474. Such Cas9 moleculesinclude Cas9 molecules of a cluster 1 bacterial family, cluster 2bacterial family, cluster 3 bacterial family, cluster 4 bacterialfamily, cluster 5 bacterial family, cluster 6 bacterial family, acluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9bacterial family, a cluster 10 bacterial family, a cluster 1 1 bacterialfamily, a cluster 12 bacterial family, a cluster 13 bacterial family, acluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16bacterial family, a cluster 17 bacterial family, a cluster 18 bacterialfamily, a cluster 19 bacterial family, a cluster 20 bacterial family, acluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23bacterial family, a cluster 24 bacterial family, a cluster 25 bacterialfamily, a cluster 26 bacterial family, a cluster 27 bacterial family, acluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30bacterial family, a cluster 31 bacterial family, a cluster 32 bacterialfamily, a cluster 33 bacterial family, a cluster 34 bacterial family, acluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37bacterial family, a cluster 38 bacterial family, a cluster 39 bacterialfamily, a cluster 40 bacterial family, a cluster 41 bacterial family, acluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44bacterial family, a cluster 45 bacterial family, a cluster 46 bacterialfamily, a cluster 47 bacterial family, a cluster 48 bacterial family, acluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51bacterial family, a cluster 52 bacterial family, a cluster 53 bacterialfamily, a cluster 54 bacterial family, a cluster 55 bacterial family, acluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58bacterial family, a cluster 59 bacterial family, a cluster 60 bacterialfamily, a cluster 61 bacterial family, a cluster 62 bacterial family, acluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65bacterial family, a cluster 66 bacterial family, a cluster 67 bacterialfamily, a cluster 68 bacterial family, a cluster 69 bacterial family, acluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72bacterial family, a cluster 73 bacterial family, a cluster 74 bacterialfamily, a cluster 75 bacterial family, a cluster 76 bacterial family, acluster 77 bacterial family, or a cluster 78 bacterial family.

In some embodiments, the naturally occurring Cas9 polypeptide isselected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2,SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. Insome embodiments, the Cas9 protein comprises an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a Cas9 amino acid sequence describedin Chylinski et al., RNA Biology 2013 10:5, 727-737; Hou et al., PNASEarly Edition 2013, 1-6).

In some embodiments, the Cas polypeptide comprises one or more of thefollowing activities:

(a) a nickase activity, i.e., the ability to cleave a single strand,e.g., the non-complementary strand or the complementary strand, of anucleic acid molecule;

(b) a double stranded nuclease activity, i.e., the ability to cleaveboth strands of a double stranded nucleic acid and create a doublestranded break, which in an embodiment is the presence of two nickaseactivities;

(c) an endonuclease activity;

(d) an exonuclease activity; and/or

(e) a helicase activity, i.e., the ability to unwind the helicalstructure of a double stranded nucleic acid.

In some embodiments, the Cas polypeptide is fused to heterologousproteins that recruit DNA-damage signaling proteins, exonucleases, orphosphatases to further increase the likelihood or the rate of repair ofthe target sequence by one repair mechanism or another. In someembodiments, a WT Cas polypeptide is co-expressed with a nucleic acidrepair template to facilitate the incorporation of an exogenous nucleicacid sequence by homology-directed repair.

In some embodiments, different Cas proteins (i.e., Cas9 proteins fromvarious species) may be advantageous to use in the various providedmethods in order to capitalize on various enzymatic characteristics ofthe different Cas proteins (e.g., for different PAM sequencepreferences; for increased or decreased enzymatic activity; for anincreased or decreased level of cellular toxicity; to change the balancebetween NHEJ, homology-directed repair, single strand breaks, doublestrand breaks, etc.).

In some embodiments, the Cas protein is a Cas9 protein derived from S.pyogenes and recognizes the PAM sequence motif NGG, NAG, NGA (Mali etal, Science 2013; 339(6121): 823-826). In some embodiments, the Casprotein is a Cas9 protein derived from S. thermophiles and recognizesthe PAM sequence motif NGGNG and/or NNAGAAW (W=A or T) (See, e.g.,Horvath et al, Science, 2010; 327(5962): 167-170, and Deveau et al, JBacteriol 2008; 190(4): 1390-1400). In some embodiments, the Cas proteinis a Cas9 protein derived from S. mutans and recognizes the PAM sequencemotif NGG and/or NAAR (R=A or G) (See, e.g., Deveau et al, J BACTERIOL2008; 190(4): 1390-1400). In some embodiments, the Cas protein is a Cas9protein derived from S. aureus and recognizes the PAM sequence motifNNGRR (R=A or G). In some embodiments, the Cas protein is a Cas9 proteinderived from S. aureus and recognizes the PAM sequence motif N GRRT (R=Aor G). In some embodiments, the Cas protein is a Cas9 protein derivedfrom S. aureus and recognizes the PAM sequence motif N GRRV (R=A or G).In some embodiments, the Cas protein is a Cas9 protein derived from N.meningitidis and recognizes the PAM sequence motif N GATT or N GCTT (R=Aor G, V=A, G or C) (See, e.g., Hou et ah, PNAS 2013, 1-6). In theaforementioned embodiments, N can be any nucleotide residue, e.g., anyof A, G, C or T. In some embodiments, the Cas protein is a Cas13aprotein derived from Leptotrichia shahii and recognizes the PFS sequencemotif of a single 3′ A, U, or C.

In some embodiments, a polynucleotide encoding a Cas protein isprovided. In some embodiments, the polynucleotide encodes a Cas proteinthat is at least 90% identical to a Cas protein described inInternational PCT Publication No. WO 2015/071474 or Chylinski et al.,RNA Biology 2013 10:5, 727-737. In some embodiments, the polynucleotideencodes a Cas protein that is at least 95%, 96%, 97%, 98%, or 99%identical to a Cas protein described in International PCT PublicationNo. WO 2015/071474 or Chylinski et al., RNA Biology 2013 10:5, 727-737.In some embodiments, the polynucleotide encodes a Cas protein that is100% identical to a Cas protein described in International PCTPublication No. WO 2015/071474 or Chylinski et al., RNA Biology 201310:5, 727-737.

2. Cas Mutants

In some embodiments, the Cas polypeptides are engineered to alter one ormore properties of the Cas polypeptide. For example, in someembodiments, the Cas polypeptide comprises altered enzymatic properties,e.g., altered nuclease activity, (as compared with a naturally occurringor other reference Cas molecule) or altered helicase activity. In someembodiments, an engineered Cas polypeptide can have an alteration thatalters its size, e.g., a deletion of amino acid sequence that reducesits size without significant effect on another property of the Caspolypeptide. In some embodiments, an engineered Cas polypeptidecomprises an alteration that affects PAM recognition. For example, anengineered Cas polypeptide can be altered to recognize a PAM sequenceother than the PAM sequence recognized by the corresponding wild-typeCas protein.

Cas polypeptides with desired properties can be made in a number ofways, including alteration of a naturally occurring Cas polypeptide orparental Cas polypeptide, to provide a mutant or altered Cas polypeptidehaving a desired property. For example, one or more mutations can beintroduced into the sequence of a parental Cas polypeptide (e.g., anaturally occurring or engineered Cas polypeptide). Such mutations anddifferences may comprise substitutions (e.g., conservative substitutionsor substitutions of non-essential amino acids); insertions; ordeletions. In some embodiments, a mutant Cas polypeptide comprises oneor more mutations (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or50 mutations) relative to a parental Cas polypeptide.

In an embodiment, a mutant Cas polypeptide comprises a cleavage propertythat differs from a naturally occurring Cas polypeptide. In someembodiments, the Cas is a deactivated Cas (dCas) mutant. In suchembodiments, the Cas polypeptide does not comprise any intrinsicenzymatic activity and is unable to mediate target nucleic acidcleavage. In such embodiments, the dCas may be fused with a heterologousprotein that is capable of modifying the target nucleic acid in anon-cleavage based manner. For example, in some embodiments, a dCasprotein is fused to transcription activator or transcription repressordomains (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3interaction domain (SID or SID4X); the ERF repressor domain (ERD); theMAX-interacting protein 1 (MXI1); methyl-CpG binding protein 2 (MECP2);etc.). In some such cases, the dCas fusion protein is targeted by theggRNA to a specific location (i.e., sequence) in the target nucleic acidand exerts locus-specific regulation such as blocking RNA polymerasebinding to a promoter (which selectively inhibits transcriptionactivator function), and/or modifying the local chromatin status (e.g.,when a fusion sequence is used that modifies the target DNA or modifiesa polypeptide associated with the target DNA). In some cases, thechanges are transient (e.g., transcription repression or activation). Insome cases, the changes are inheritable (e.g., when epigeneticmodifications are made to the target DNA or to proteins associated withthe target DNA, e.g., nucleosomal histones).

In some embodiments, the dCas is a dCas13 mutant (Konermann et al., Cell173 (2018), 665-676). These dCas13 mutants can then be fused to enzymesthat modify RNA, including adenosine deaminases (e.g., ADAR1 and ADAR2).Adenosine deaminases convert adenine to inosine, which the translationalmachinery treats like guanine, thereby creating a functional A→G changein the RNA sequence. In some embodiments, the dCas is a dCas9 mutant.

In some embodiments, the mutant Cas9 is a Cas9 nickase mutant. Cas9nickase mutants comprise only one catalytically active domain (eitherthe HNH domain or the RuvC domain). The Cas9 nickase mutants retain DNAbinding based on gRNA specificity, but are capable of cutting only onestrand of DNA resulting in a single-strand break (e.g. a “nick”). Insome embodiments, two complementary Cas9 nickase mutants (e.g., one Cas9nickase mutant with an inactivated RuvC domain, and one Cas9 nickasemutant with an inactivated HNH domain) are expressed in the same cellwith two gRNAs corresponding to two respective target sequences; onetarget sequence on the sense DNA strand, and one on the antisense DNAstrand. This dual-nickase system results in staggered double strandedbreaks and can increase target specificity, as it is unlikely that twooff-target nicks will be generated close enough to generate a doublestranded break. In some embodiments, a Cas9 nickase mutant isco-expressed with a nucleic acid repair template to facilitate theincorporation of an exogenous nucleic acid sequence by homology-directedrepair.

In some embodiments, the Cas polypeptides described herein can beengineered to alter the PAM/PFS specificity of the Cas polypeptide. Insome embodiments, a mutant Cas polypeptide has a PAM/PFS specificitythat is different from the PAM/PFS specificity of the parental Caspolypeptide. For example, a naturally occurring Cas protein can bemodified to alter the PAM/PFS sequence that the mutant Cas polypeptiderecognizes to decrease off target sites, improve specificity, oreliminate a PAM/PFS recognition requirement. In some embodiments, a Casprotein can be modified to increase the length of the PAM/PFSrecognition sequence. In some embodiments, the length of the PAMrecognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acidsin length. Cas polypeptides that recognize different PAM/PFS sequencesand/or have reduced off-target activity can be generated using directedevolution. Exemplary methods and systems that can be used for directedevolution of Cas polypeptides are described, e.g., in Esvelt et al.Nature 2011, 472(7344): 499-503.

Exemplary Cas mutants are described in International PCT Publication No.WO 2015/161276 and Konermann et al., Cell 173 (2018), 665-676 which areincorporated herein by reference in their entireties.

3. gRNAs

The present disclosure provides guide RNAs (gRNAs) that direct asite-directed modifying polypeptide to a specific target nucleic acidsequence. A gRNA comprises a “nucleic acid-targeting domain” or“targeting domain” and protein-binding segment. The targeting domain mayalso be referred to as a “spacer” sequence and comprises a nucleotidesequence that is complementary to a target nucleic acid sequence. Assuch, the targeting domain segment of a gRNA interacts with a targetnucleic acid in a sequence-specific manner via hybridization (i.e., basepairing) and determines the location within the target nucleic acid thatthe gRNA will bind. The targeting domain segment of a gRNA can bemodified (e.g., by genetic engineering) to hybridize to a desiredsequence within a target nucleic acid sequence. In some embodiments, thetargeting domain sequence is between about 13 and about 22 nucleotidesin length. In some embodiments, the targeting domain sequence is about13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In someembodiments, the targeting domain sequence is about 20 nucleotides inlength.

The protein-binding segment of a gRNA interacts with a site-directedmodifying polypeptide (e.g. a Cas protein) to form a ribonucleoprotein(RNP) complex comprising the gRNA and the site-directed modifyingpolypeptide. The targeting domain segment of the gRNA then guides thebound site-directed modifying polypeptide to a specific nucleotidesequence within target nucleic acid via the above-described spacersequence. The protein-binding segment of a gRNA comprises at least twostretches of nucleotides that are complementary to one another and whichform a double stranded RNA duplex. The protein-binding segment of a gRNAmay also be referred to as a “scaffold” segment or a “tracr RNA”. Insome embodiments, the tracr RNA sequence is between about 30 and about180 nucleotides in length. In some embodiments, the tracr RNA sequenceis between about 40 and about 90 nucleotides, about 50 and about 90nucleotides, about 60 and about 90 nucleotides, about 65 and about 85nucleotides, about 70 and about 80 nucleotides, about 65 and about 75nucleotides, or about 75 and about 85 nucleotides in length. In someembodiments, the tracr RNA sequence is about 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or about 90 nucleotidesin length. In some embodiments, the tracr RNA comprises a nucleic acidsequence encoded by the DNA sequence of SEQ ID NO: 34 (See Mali et al.,Science (2013) 339(6121):823-826), SEQ ID NOs: 35-36 (See PCTPublication No. WO 2016/106236), SEQ ID NOs: 37-39 (See Deltcheva etal., Nature. 2011 Mar. 31; 471(7340): 602-607), or SEQ ID NO: 40 (SeeChen et al., Cell. 2013; 155(7); 1479-1491). Any of the foregoing tracrsequences are suitable for use in combination with any of the gRNAtargeting domain embodiments described herein.

In some embodiments, a gRNA comprises two separate RNA molecules (i.e.,a “dual gRNA”). In some embodiments, a gRNA comprises a single RNAmolecule (i.e. a “single guide RNA” or “sgRNA”). Herein, use of the term“guide RNA” or “gRNA” is inclusive of both dual gRNAs and sgRNAs. A dualgRNA comprises two separate RNA molecules: a “crispr RNA” (or “crRNA”)and a “tracr RNA”. A crRNA molecule comprises a spacer sequencecovalently linked to a “tracr mate” sequence. The tracer mate sequencecomprises a stretch of nucleotides that are complementary to acorresponding sequence in the tracr RNA molecule. The crRNA molecule andtracr RNA molecule hybridize to one another via the complementarity ofthe tracr and tracer mate sequences.

In some embodiments, the gRNA is an sgRNA. In such embodiments, thenucleic acid-targeting sequence and the protein-binding sequence arepresent in a single RNA molecule by fusion of the spacer sequence to thetracr RNA sequence. In some embodiments, the sgRNA is about 50 to about200 nucleotides in length. In some embodiments, the sgRNA is about 75 toabout 150 or about 100 to about 125 nucleotides in length. In someembodiments, the sgRNA is about 100 nucleotides in length.

In some embodiments, the gRNAs of the present disclosure comprise atargeting domain sequence that is least 90%, 95%, 96%, 97%, 98%, or 99%complementary, or is 100% complementary to a target nucleic acidsequence within a target locus. In some embodiments, the target nucleicacid sequence is an RNA target sequence. In some embodiments, the targetnucleic acid sequence is a DNA target sequence.

In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to asequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR (e.g., a gene selected from Table 2). In someembodiments, the targeting domain sequence binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B. In some embodiments, thetargeting domain sequence binds to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toone of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813. In some embodiments,the gRNAs provided herein comprise a targeting domain sequence thatbinds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to a target DNA sequence of theCBLB gene. In some embodiments, the nucleic acid-binding segments of thegRNA sequences bind to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQ IDNOs: 499-524. Additional gRNAs suitable for targeting CBLB are describedin US Patent Application Publication No. 2017/0175128.

In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of the TNFAIP3 gene. In some embodiments, thenucleic acid-binding segments of the gRNA sequences bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 348-396 or SEQ ID NOs: 348-3486.In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of the BCOR gene. In some embodiments, the nucleicacid-binding segments of the gRNA sequences bind to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 708-772 or SEQ ID NOs: 708-764.

In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to asequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS,ZC3H12A, MAP4K1, and NR4A3 (e.g., a gene selected from Table 3). In someembodiments, the targeting domain sequence binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Tables 6A-Table 6H. In some embodiments, thetargeting domain sequence binds to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toone of SEQ ID NOs: 814-1566.

In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to asequence of a target gene selected from BCL2L11, FLI1, CALM2, DHODH,UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, andGNAS. In some embodiments, the targeting domain sequence binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to a target DNA sequence defined by aset of genomic coordinates shown in Table 6A or Table 6B. In someembodiments, the targeting domain sequence binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 814-1064. In some embodiments,the gRNAs provided herein comprise a targeting domain sequence thatbinds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to a sequence of ZC3H12A. In someembodiments, the targeting domain sequence binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 6C or Table 6D. In some embodiments, thetargeting domain sequence binds to a target DNA sequence that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical toone of SEQ ID NOs: 1065-1509. In some embodiments, the targeting domainsequence binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1065-1264.

In some embodiments, the gRNAs provided herein comprise a targetingdomain sequence that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to asequence of the MAP4K1 gene. In some embodiments, the targeting domainsequence binds to a target DNA sequence that is at least 90%, 95%, 96%,97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 6E orTable 6F. In some embodiments, the targeting domain sequence binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 510-1538. In someembodiments, the gRNAs provided herein comprise a targeting domainsequence that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a sequence ofthe NR4A3 gene. In some embodiments, the targeting domain sequence bindsto a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or99% identical, or is 100% identical to a target DNA sequence defined bya set of genomic coordinates shown in Table 6G or Table 6H. In someembodiments, the targeting domain sequence binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 1539-1566.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR (e.g., a gene selected from Table 2) and wherein at leastone of the gRNAs comprises a targeting domain that binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence of a target gene selectedfrom BCL2L11, FLI1, CALM2, DHODH, (IMPS, RBM39, SEMA7A, CHIC2, PCBP1,PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and NR4A3 (e.g., agene selected from Table 3).

In some embodiments, at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in Table 5A orTable 5B and at least one of the gRNAs comprises a targeting domain thatbinds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to a target DNA sequence definedby a set of genomic coordinates shown in one of Tables 6A-6H. In someembodiments, at least one of the gRNAs comprises a targeting domain thatbinds to a target DNA sequence that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical, or is 100% identical to one of SEQ ID NOs: 154-498 orSEQ ID NOs: 499-813 and at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to one of SEQ IDNOs: 814-1566. In some embodiments, at least one of the gRNAs comprisesa targeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813 and at least one of thegRNAs comprises a targeting domain encoded by a nucleic acid sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 814-1566.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAGS, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and wherein at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of a target gene selected from BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,and GNAS. In some embodiments, at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence defined by a set of genomic coordinates shown inTable 5A or Table 5B and at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence defined by a set of genomic coordinates shown in one of Table6A or Table 6B. In some embodiments, at least one of the gRNAs comprisesa targeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813 and at least one of thegRNAs comprises a targeting domain that binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 814-1064. In some embodiments, at leastone of the gRNAs comprises a targeting domain encoded by a nucleic acidsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813and at least one of the gRNAs comprises a targeting domain encoded by anucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 814-1064.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of the CBLB and wherein at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of a target gene selected from BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,and GNAS. In some embodiments, at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 499-524 and at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 814-1064. In some embodiments, at least one of the gRNAscomprises a targeting domain encoded by a nucleic acid sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto one of SEQ ID NOs: 499-524 and at least one of the gRNAs comprises atargeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 814-1064.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and wherein at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of ZC3H12A. In some embodiments, at least one of thegRNAs comprises a targeting domain that binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B and at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence defined by a set of genomic coordinates shownin one of Table 6C or Table 6D. In some embodiments, at least one of thegRNAs comprises a targeting domain that binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1065-1509. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:154-498 or SEQ ID NOs: 499-813 and at least one of the gRNAs comprises atargeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1509.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of the CBLB gene and wherein at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence of the ZC3H12A gene. In some embodiments, atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 499-524 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1065-1509. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524 and at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1065-1509. In some embodiments, at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 499-524 and at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1264. In some embodiments, at least one of the gRNAscomprises a targeting domain encoded by a nucleic acid sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto one of SEQ ID NOs: 499-524 and at least one of the gRNAs comprises atargeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1065-1264.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and wherein at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of the MAP4K1 gene. In some embodiments, at leastone of the gRNAs comprises a targeting domain that binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B and at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence defined by a set of genomic coordinates shownin one of Table 6E or Table 6F. In some embodiments, at least one of thegRNAs comprises a targeting domain that binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1510-1538. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:154-498 or SEQ ID NOs: 499-813 and at least one of the gRNAs comprises atargeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1510-1538.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of the CBLB gene and wherein at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence of the MAP4K1 gene. In some embodiments, atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 499-524 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1510-1538. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524 and at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1510-1538.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of a target gene selected from IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, or BCOR and wherein at least one of the gRNAs comprises atargeting domain that binds to a target DNA sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to atarget DNA sequence of the NR4A3 gene. In some embodiments, at least oneof the gRNAs comprises a targeting domain that binds to a target DNAsequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, oris 100% identical to a target DNA sequence defined by a set of genomiccoordinates shown in Table 5A or Table 5B and at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence defined by a set of genomic coordinates shownin one of Table 6G or Table 6H. In some embodiments, at least one of thegRNAs comprises a targeting domain that binds to a target DNA sequencethat is at least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical to one of SEQ ID NOs: 154-498 or SEQ ID NOs: 499-813 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1539-1566. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:154-498 or SEQ ID NOs: 499-813 and at least one of the gRNAs comprises atargeting domain encoded by a nucleic acid sequence that is at least90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical to oneof SEQ ID NOs: 1539-1566.

In some embodiments, the gene-regulating system comprises two or moregRNA molecules, wherein at least one of the gRNAs comprises a targetingdomain that binds to a target DNA sequence that is at least 90%, 95%,96%, 97%, 98%, or 99% identical, or is 100% identical to a target DNAsequence of the CBLB gene and wherein at least one of the gRNAscomprises a targeting domain that binds to a target DNA sequence that isat least 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identicalto a target DNA sequence of the NR4A3 gene. In some embodiments, atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 499-524 and atleast one of the gRNAs comprises a targeting domain that binds to atarget DNA sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical, or is 100% identical to one of SEQ ID NOs: 1539-1566. In someembodiments, at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:499-524 and at least one of the gRNAs comprises a targeting domainencoded by a nucleic acid sequence that is at least 90%, 95%, 96%, 97%,98%, or 99% identical, or is 100% identical to one of SEQ ID NOs:1539-1566.

In some embodiments, the nucleic acid-binding segments of the gRNAsequences described herein are designed to minimize off-target bindingusing algorithms known in the art (e.g., Cas-OFF finder) to identifytarget sequences that are unique to a particular target locus or targetgene.

In some embodiments, the gRNAs described herein can comprise one or moremodified nucleosides or nucleotides which introduce stability towardnucleases. In such embodiments, these modified gRNAs may elicit areduced innate immune as compared to a non-modified gRNA. The term“innate immune response” includes a cellular response to exogenousnucleic acids, including single stranded nucleic acids, generally ofviral or bacterial origin, which involves the induction of cytokineexpression and release, particularly the interferons, and cell death.

In some embodiments, the gRNAs described herein are modified at or nearthe 5′ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of their 5′ end).In some embodiments, the 5′ end of a gRNA is modified by the inclusionof a eukaryotic mRNA cap structure or cap analog (e.g., a G(5′)ppp(5′)Gcap analog, a m7G(5′)ppp(5′)G cap analog, or a 3′-0-Me-m7G(5′)ppp(5′)Ganti reverse cap analog (ARCA)). In some embodiments, an in vitrotranscribed gRNA is modified by treatment with a phosphatase (e.g., calfintestinal alkaline phosphatase) to remove the 5′ triphosphate group. Insome embodiments, a gRNA comprises a modification at or near its 3′ end(e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3′ end). For example,in some embodiments, the 3′ end of a gRNA is modified by the addition ofone or more (e.g., 25-200) adenine (A) residues.

In some embodiments, modified nucleosides and modified nucleotides canbe present in a gRNA, but also may be present in other gene-regulatingsystems, e.g., mRNA, RNAi, or siRNA-based systems. In some embodiments,modified nucleosides and nucleotides can include one or more of:

(a) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester backbone linkage;

(b) alteration, e.g., replacement, of a constituent of the ribose sugar,e.g., of the 2′ hydroxyl on the ribose sugar;

(c) wholesale replacement of the phosphate moiety with “dephospho”linkers;

(d) modification or replacement of a naturally occurring nucleobase;

(e) replacement or modification of the ribose-phosphate backbone;

(f) modification of the 3′ end or 5′ end of the oligonucleotide, e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety; and

(g) modification of the sugar.

In some embodiments, the modifications listed above can be combined toprovide modified nucleosides and nucleotides that can have two, three,four, or more modifications. For example, in some embodiments, amodified nucleoside or nucleotide can have a modified sugar and amodified nucleobase. In some embodiments, every base of a gRNA ismodified. In some embodiments, each of the phosphate groups of a gRNAmolecule are replaced with phosphorothioate groups.

In some embodiments, a software tool can be used to optimize the choiceof gRNA within a user's target sequence, e.g., to minimize totaloff-target activity across the genome. Off target activity may be otherthan cleavage. For example, for each possible gRNA choice using S.pyogenes Cas9, software tools can identify all potential off-targetsequences (preceding either NAG or NGG PAMs) across the genome thatcontain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)of mismatched base-pairs. The cleavage efficiency at each off-targetsequence can be predicted, e.g., using an experimentally-derivedweighting scheme. Each possible gRNA can then be ranked according to itstotal predicted off-target cleavage; the top-ranked gRNAs representthose that are likely to have the greatest on-target and the leastoff-target cleavage. Other functions, e.g., automated reagent design forgRNA vector construction, primer design for the on-target Surveyorassay, and primer design for high-throughput detection andquantification of off-target cleavage via next-generation sequencing,can also be included in the tool.

IV. Polynucleotides

In some embodiments, the present disclosure provides polynucleotides ornucleic acid molecules encoding a gene-regulating system describedherein. As used herein, the terms “nucleotide” or “nucleic acid” referto deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNAhybrids. Polynucleotides may be single-stranded or double-stranded andeither recombinant, synthetic, or isolated. Polynucleotides include, butare not limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA),RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA),synthetic DNA, or recombinant DNA. Polynucleotides refer to a polymericform of nucleotides of at least 5, at least 10, at least 15, at least20, at least 25, at least 30, at least 40, at least 50, at least 100, atleast 200, at least 300, at least 400, at least 500, at least 1000, atleast 5000, at least 10000, or at least 15000 or more nucleotides inlength, either ribonucleotides or deoxyribonucleotides or a modifiedform of either type of nucleotide, as well as all intermediate lengths.It will be readily understood that “intermediate lengths,” in thiscontext, means any length between the quoted values, such as 6, 7, 8, 9,etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

In particular embodiments, polynucleotides may be codon-optimized. Asused herein, the term “codon-optimized” refers to substituting codons ina polynucleotide encoding a polypeptide in order to increase theexpression, stability and/or activity of the polypeptide. Factors thatinfluence codon optimization include, but are not limited to one or moreof: (i) variation of codon biases between two or more organisms or genesor synthetically constructed bias tables, (ii) variation in the degreeof codon bias within an organism, gene, or set of genes, (iii)systematic variation of codons including context, (iv) variation ofcodons according to their decoding tRNAs, (v) variation of codonsaccording to GC %, either overall or in one position of the triplet,(vi) variation in degree of similarity to a reference sequence forexample a naturally occurring sequence, (vii) variation in the codonfrequency cutoff, (viii) structural properties of mRNAs transcribed fromthe DNA sequence, (ix) prior knowledge about the function of the DNAsequences upon which design of the codon substitution set is to bebased, (x) systematic variation of codon sets for each amino acid, (xi)isolated removal of spurious translation initiation sites and/or (xii)elimination of fortuitous polyadenylation sites otherwise leading totruncated RNA transcripts.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. A “comparisonwindow” refers to a conceptual segment of at least 6 contiguouspositions, usually about 50 to about 100, more usually about 100 toabout 150 in which a sequence is compared to a reference sequence of thesame number of contiguous positions after the two sequences areoptimally aligned. Thus, a “percentage of sequence identity” may becalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T, C, G, I) or the identical aminoacid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr,Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity.

As used herein, the terms “polynucleotide variant” and “variant” and thelike refer to polynucleotides displaying substantial sequence identitywith a reference polynucleotide sequence or polynucleotides thathybridize with a reference sequence under stringent conditions that aredefined hereinafter. These terms include polynucleotides in which one ormore nucleotides have been added or deleted, or replaced with differentnucleotides compared to a reference polynucleotide. In this regard, itis well understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains thebiological function or activity of the reference polynucleotide.

In particular embodiments, polynucleotides or variants have at least orabout 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to areference sequence.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide, or fragment of variantthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated in particular embodiments, for examplepolynucleotides that are optimized for human and/or primate codonselection. Further, alleles of the genes comprising the polynucleotidesequences provided herein may also be used. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art, such that theiroverall length may vary considerably. It is therefore contemplated thata polynucleotide fragment of almost any length may be employed inparticular embodiments, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

Polynucleotides can be prepared, manipulated and/or expressed using anyof a variety of well-established techniques known and available in theart.

Vectors

In order to express a gene-regulating system described herein in a cell,an expression cassette encoding the gene-regulating system can beinserted into appropriate vector. The term “nucleic acid vector” is usedherein to refer to a nucleic acid molecule capable transferring ortransporting another nucleic acid molecule. The transferred nucleic acidis generally linked to, e.g., inserted into, the vector nucleic acidmolecule. A nucleic acid vector may include sequences that directautonomous replication in a cell, or may include sequences sufficient toallow integration into host cell DNA.

The term “expression cassette” as used herein refers to geneticsequences within a vector which can express an RNA, and subsequently aprotein. The nucleic acid cassette contains the gene of interest, e.g.,a gene-regulating system. The nucleic acid cassette is positionally andsequentially oriented within the vector such that the nucleic acid inthe cassette can be transcribed into RNA, and when necessary, translatedinto a protein or a polypeptide, undergo appropriate post-translationalmodifications required for activity in the transformed cell, and betranslocated to the appropriate compartment for biological activity bytargeting to appropriate intracellular compartments or secretion intoextracellular compartments. Preferably, the cassette has its 3′ and 5′ends adapted for ready insertion into a vector, e.g., it has restrictionendonuclease sites at each end. The cassette can be removed and insertedinto a plasmid or viral vector as a single unit.

In particular embodiments, vectors include, without limitation,plasmids, phagemids, cosmids, transposons, artificial chromosomes suchas yeast artificial chromosome (YAC), bacterial artificial chromosome(BAC), or P1-derived artificial chromosome (PAC), bacteriophages such aslambda phage or M13 phage, and animal viruses. In particularembodiments, the coding sequences of the gene-regulating systemsdisclosed herein can be ligated into such vectors for the expression ofthe gene-regulating systems in mammalian cells.

In some embodiments, non-viral vectors are used to deliver one or morepolynucleotides contemplated herein to an immune effector cell, e.g., aT cell. In some embodiments, the recombinant vector comprising apolynucleotide encoding one or more components of a gene-regulatingsystem described herein is a plasmid. Numerous suitable plasmidexpression vectors are known to those of skill in the art, and many arecommercially available. The following vectors are provided by way ofexample; for eukaryotic host cells: pXT1, pSG5 (Stratagene), pSVK3,pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other plasmid vectormay be used so long as it is compatible with the host cell. Depending onthe cell type and gene-regulating system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, the recombinant vector comprising a polynucleotideencoding one or more components of a gene-regulating system describedherein is a viral vector. Suitable viral vectors include, but are notlimited to, viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549,1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., U.S. Pat. No. 7,078,387;Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al, PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastavain WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelsonet al, Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993)90:10613-10617); SV40; herpes simplex virus; human immunodeficiencyvirus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi etal., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., MurineLeukemia Virus, spleen necrosis virus, and vectors derived fromretroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, a lentivirus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus); and thelike. Examples of vectors are pClneo vectors (Promega) for expression inmammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™, andpLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transferand expression in mammalian cells.

In some embodiments, the vector is a non-integrating vector, includingbut not limited to, an episomal vector or a vector that is maintainedextrachromosomally. As used herein, the term “episomal” refers to avector that is able to replicate without integration into host'schromosomal DNA and without gradual loss from a dividing host cell alsomeaning that said vector replicates extrachromosomally or episomally.The vector is engineered to harbor the sequence coding for the origin ofDNA replication or “ori” from a lymphotrophic herpes virus or a gammaherpesvirus, an adenovirus, SV40, a bovine papilloma virus, or a yeast,specifically a replication origin of a lymphotrophic herpes virus or agamma herpesvirus corresponding to oriP of EBV. In a particular aspect,the lymphotrophic herpes virus may be Epstein Barr virus (EBV), Kaposi'ssarcoma herpes virus (KSHV), Herpes virus saimiri (HS), or Marek'sdisease virus (MDV). Epstein Barr virus (EBV) and Kaposi's sarcomaherpes virus (KSHV) are also examples of a gamma herpesvirus.

In some embodiments, a polynucleotide is introduced into a target orhost cell using a transposon vector system. In certain embodiments, thetransposon vector system comprises a vector comprising transposableelements and a polynucleotide contemplated herein; and a transposase. Inone embodiment, the transposon vector system is a single transposasevector system, see, e.g., WO 2008/027384. Exemplary transposasesinclude, but are not limited to: piggyBac, Sleeping Beauty, Mosl,Tcl/mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, andderivatives thereof. The piggyBac transposon and transposase aredescribed, for example, in U.S. Pat. No. 6,962,810, which isincorporated herein by reference in its entirety. The Sleeping Beautytransposon and transposase are described, for example, in Izsvak et al.,J. Mol. Biol. 302: 93-102 (2000), which is incorporated herein byreference in its entirety. The Tol2 transposon which was first isolatedfrom the medaka fish Oryzias latipes and belongs to the hAT family oftransposons is described in Kawakami et al. (2000). Mini-Tol2 is avariant of Tol2 and is described in Balciunas et al. (2006). The Tol2and Mini-Tol2 transposons facilitate integration of a transgene into thegenome of an organism when co-acting with the Tol2 transposase. The FrogPrince transposon and transposase are described, for example, in Miskeyet al., Nucleic Acids Res. 31:6873-6881 (2003).

In some embodiments, a polynucleotide sequence encoding one or morecomponents of a gene-regulating system described herein is operablylinked to a control element, e.g., a transcriptional control element,such as a promoter. “Control elements” refer those non-translatedregions of the vector (e.g., origin of replication, selection cassettes,promoters, enhancers, translation initiation signals (Shine Dalgarnosequence or Kozak sequence) introns, a polyadenylation sequence, 5′ and3′ untranslated regions) which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. The transcriptional control element may befunctional in either a eukaryotic cell (e.g., a mammalian cell) or aprokaryotic cell (e.g., bacterial or archaeal cell). In someembodiments, a polynucleotide sequence encoding one or more componentsof a gene-regulating system described herein is operably linked tomultiple control elements that allow expression of the polynucleotide inboth prokaryotic and eukaryotic cells.

Depending on the cell type and gene-regulating system utilized, any of anumber of suitable transcription and translation control elements,including constitutive and inducible promoters, transcription enhancerelements, transcription terminators, etc. may be used in the expressionvector (see e.g., Bitter et al. (1987) Methods in Enzymology,153:516-544). The term “promoter” as used herein refers to a recognitionsite of a polynucleotide (DNA or RNA) to which an RNA polymerase binds.An RNA polymerase initiates and transcribes polynucleotides operablylinked to the promoter. In particular embodiments, promoters operativein mammalian cells comprise an AT-rich region located approximately 25to 30 bases upstream from the site where transcription is initiatedand/or another sequence found 70 to 80 bases upstream from the start oftranscription, a CNCAAT region where N may be any nucleotide. The term“enhancer” refers to a segment of DNA which contains sequences capableof providing enhanced transcription and in some instances can functionindependent of their orientation relative to another control sequence.An enhancer can function cooperatively or additively with promotersand/or other enhancer elements.

In some embodiments, polynucleotides encoding one or more components ofa gene-regulating system described herein are operably linked to apromoter. The term “operably linked”, refers to a juxtaposition whereinthe components described are in a relationship permitting them tofunction in their intended manner. In one embodiment, the term refers toa functional linkage between a nucleic acid expression control sequence(such as a promoter, and/or enhancer) and a second polynucleotidesequence, e.g., a polynucleotide encoding one or more components of agene-regulating system, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, aviral simian virus 40 (SV40) (e.g., early and late SV40), a spleen focusforming virus (SFFV) promoter, long terminal repeats (LTRs) fromretrovirus (e.g., a Moloney murine leukemia virus (MoMLV) LTR promoteror a Rous sarcoma virus (RSV) LTR), a herpes simplex virus (HSV)(thymidine kinase) promoter, H5, P7.5, and P11 promoters from vacciniavirus, an elongation factor 1-alpha (EF1α) promoter, early growthresponse 1 (EGR1) promoter, a ferritin H (FerH) promoter, a ferritin L(FerL) promoter, a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)promoter, a eukaryotic translation initiation factor 4A1 (EIF4A1)promoter, a heat shock 70 kDa protein 5 (HSPA5) promoter, a heat shockprotein 90 kDa beta, member 1 (HSP90B1) promoter, a heat shock protein70 kDa (HSP70) promoter, a β-kinesin (β-KIN) promoter, the human ROSA 26locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), aUbiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter,a cytomegalovirus enhancer/chicken β-actin (CAG) promoter, a β-actinpromoter and a myeloproliferative sarcoma virus enhancer, negativecontrol region deleted, d1587rev primer-binding site substituted (MND)promoter, and mouse metallothionein-1. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector may also include appropriate sequences for amplifying expression.The expression vector may also include nucleotide sequences encodingprotein tags (e.g., 6×His tag, hemagglutinin tag, green fluorescentprotein, etc.) that are fused to the site-directed modifyingpolypeptide, thus resulting in a chimeric polypeptide.

In some embodiments, a polynucleotide sequence encoding one or morecomponents of a gene-regulating system described herein is operablylinked to a constitutive promoter. In such embodiments, thepolynucleotides encoding one or more components of a gene-regulatingsystem described herein are constitutively and/or ubiquitously expressedin a cell.

In some embodiments, a polynucleotide sequence encoding one or morecomponents of a gene-regulating system described herein is operablylinked to an inducible promoter. In such embodiments, polynucleotidesencoding one or more components of a gene-regulating system describedherein are conditionally expressed. As used herein, “conditionalexpression” may refer to any type of conditional expression including,but not limited to, inducible expression; repressible expression;expression in cells or tissues having a particular physiological,biological, or disease state (e.g., cell type or tissue specificexpression) etc. Illustrative examples of inducible promoters/systemsinclude, but are not limited to, steroid-inducible promoters such aspromoters for genes encoding glucocorticoid or estrogen receptors(inducible by treatment with the corresponding hormone), metallothioninepromoter (inducible by treatment with various heavy metals), MX-1promoter (inducible by interferon), the “GeneSwitch”mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), thecumate inducible gene switch (WO 2002/088346), tetracycline-dependentregulatory systems, etc.

In some embodiments, the vectors described herein further comprise atranscription termination signal. Elements directing the efficienttermination and polyadenylation of the heterologous nucleic acidtranscripts increases heterologous gene expression. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal. In particular embodiments, vectors comprise apolyadenylation sequence 3′ of a polynucleotide encoding a polypeptideto be expressed. The term “polyA site” or “polyA sequence” as usedherein denotes a DNA sequence which directs both the termination andpolyadenylation of the nascent RNA transcript by RNA polymerase II.Polyadenylation sequences can promote mRNA stability by addition of apolyA tail to the 3′ end of the coding sequence and thus, contribute toincreased translational efficiency. Cleavage and polyadenylation isdirected by a poly(A) sequence in the RNA. The core poly(A) sequence formammalian pre-mRNAs has two recognition elements flanking acleavage-polyadenylation site. Typically, an almost invariant AAUAAAhexamer lies 20-50 nucleotides upstream of a more variable element richin U or GU residues. Cleavage of the nascent transcript occurs betweenthese two elements and is coupled to the addition of up to 250adenosines to the 5′ cleavage product. In particular embodiments, thecore poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA,AGTAAA). In particular embodiments, the poly(A) sequence is an SV40polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbitβ-globin polyA sequence (rβgpA), variants thereof, or another suitableheterologous or endogenous polyA sequence known in the art.

In some embodiments, a vector may also comprise a sequence encoding asignal peptide (e.g., for nuclear localization, nucleolar localization,mitochondrial localization), fused to the polynucleotide encoding theone or more components of the system. For example, a vector may comprisea nuclear localization sequence (e.g., from SV40) fused to thepolynucleotide encoding the one or more components of the system.

Methods of introducing polynucleotides and recombinant vectors into ahost cell are known in the art, and any known method can be used tointroduce components of a gene-regulating system into a cell. Suitablemethods include e.g., viral or bacteriophage infection, transfection,conjugation, protoplast fusion, lipofection, electroporation, calciumphosphate precipitation, polyethyleneimine (PEI)-mediated transfection,DEAE-dextran mediated transfection, liposome-mediated transfection,particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, e.g.,Panyam et al., Adv Drug Deliv Rev. 2012 Sep. 13. pii:50169-409X(12)00283-9), microfluidics delivery methods (See e.g.,International PCT Publication No. WO 2013/059343), and the like. In someembodiments, delivery via electroporation comprises mixing the cellswith the components of a gene-regulating system in a cartridge, chamber,or cuvette and applying one or more electrical impulses of definedduration and amplitude. In some embodiments, cells are mixed withcomponents of a gene-regulating system in a vessel connected to a device(e.g., a pump) which feeds the mixture into a cartridge, chamber, orcuvette wherein one or more electrical impulses of defined duration andamplitude are applied, after which the cells are delivered to a secondvessel. Illustrative examples of polynucleotide delivery systemssuitable for use in particular embodiments contemplated in particularembodiments include, but are not limited to, those provided by AmaxaBiosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, Neon™Transfection Systems, and Copernicus Therapeutics Inc. Lipofectionreagents are sold commercially (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficient lipofectionof polynucleotides have been described in the literature. See e.g., Liuet al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journalof Drug Delivery. 2011:1-12.

In some embodiments, vectors comprising polynucleotides encoding one ormore components of a gene-regulating system described herein areintroduced to cells by viral delivery methods, e.g., by viraltransduction. In some embodiments, vectors comprising polynucleotidesencoding one or more components of a gene-regulating system describedherein are introduced to cells by non-viral delivery methods.Illustrative methods of non-viral delivery of polynucleotidescontemplated in particular embodiments include, but are not limited to:electroporation, sonoporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, nanoparticles, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions,DEAE-dextran-mediated transfer, gene gun, and heat-shock.

In some embodiments, one or more components of a gene-regulating system,or polynucleotide sequence encoding one or more components of agene-regulating system described herein are introduced to a cell in anon-viral delivery vehicle, such as a transposon, a nanoparticle (e.g.,a lipid nanoparticle), a liposome, an exosome, an attenuated bacterium,or a virus-like particle. In some embodiments, the vehicle is anattenuated bacterium (e.g., naturally or artificially engineered to beinvasive but attenuated to prevent pathogenesis including Listeriamonocytogenes, certain Salmonella strains, Bifidobacterium longum, andmodified Escherichia coli), bacteria having nutritional andtissue-specific tropism to target specific cells, and bacteria havingmodified surface proteins to alter target cell specificity. In someembodiments, the vehicle is a genetically modified bacteriophage (e.g.,engineered phages having large packaging capacity, less immunogenicity,containing mammalian plasmid maintenance sequences and havingincorporated targeting ligands). In some embodiments, the vehicle is amammalian virus-like particle. For example, modified viral particles canbe generated (e.g., by purification of the “empty” particles followed byex vivo assembly of the virus with the desired cargo). The vehicle canalso be engineered to incorporate targeting ligands to alter targettissue specificity. In some embodiments, the vehicle is a biologicalliposome. For example, the biological liposome is a phospholipid-basedparticle derived from human cells (e.g., erythrocyte ghosts, which arered blood cells broken down into spherical structures derived from thesubject and wherein tissue targeting can be achieved by attachment ofvarious tissue or cell-specific ligands), secretory exosomes, orsubjectiderived membrane-bound nanovesicles (30-100 nm) of endocyticorigin (e.g., can be produced from various cell types and can thereforebe taken up by cells without the need for targeting ligands).

V. Methods of Producing Modified Immune Effector Cells

In some embodiments, the present disclosure provides methods forproducing modified immune effector cells. In some embodiments, themethods comprise introducing a gene-regulating system into a populationof immune effector cells wherein the gene-regulating system is capableof reducing expression and/or function of one or more endogenous targetgenes.

The components of the gene-regulating systems described herein, e.g., anucleic acid-, protein-, or nucleic acid/protein-based system can beintroduced into target cells in a variety of forms using a variety ofdelivery methods and formulations. In some embodiments, a polynucleotideencoding one or more components of the system is delivered by arecombinant vector (e.g., a viral vector or plasmid, described supra).In some embodiments, where the system comprises more than a singlecomponent, a vector may comprise a plurality of polynucleotides, eachencoding a component of the system. In some embodiments, where thesystem comprises more than a single component, a plurality of vectorsmay be used, wherein each vector comprises a polynucleotide encoding aparticular component of the system. In some embodiments, theintroduction of the gene-regulating system to the cell occurs in vitro.In some embodiments, the introduction of the gene-regulating system tothe cell occurs in vivo. In some embodiments, the introduction of thegene-regulating system to the cell occurs ex vivo.

In particular embodiments, the introduction of the gene-regulatingsystem to the cell occurs in vitro or ex vivo. In some embodiments, theimmune effector cells are modified in vitro or ex vivo without furthermanipulation in culture. For example, in some embodiments, the methodsof producing a modified immune effector cell described herein compriseintroduction of a gene-regulating system in vitro or ex vivo withoutadditional activation and/or expansion steps. In some embodiments, theimmune effector cells are modified and are further manipulated in vitroor ex vivo. For example, in some embodiments, the immune effector cellsare activated and/or expanded in vitro or ex vivo prior to introductionof a gene-regulating system. In some embodiments, a gene-regulatingsystem is introduced to the immune effector cells and are then activatedand/or expanded in vitro or ex vivo. In some embodiments, successfullymodified cells can be sorted and/or isolated (e.g., by flow cytometry)from unsuccessfully modified cells to produce a purified population ofmodified immune effector cells. These successfully modified cells canthen be further propagated to increase the number of the modified cellsand/or cryopreserved for future use.

In some embodiments, the present disclosure provides methods forproducing modified immune effector cells comprising obtaining apopulation of immune effector cells. The population of immune effectorcells may be cultured in vitro under various culture conditionsnecessary to support growth, for example, at an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂) and in anappropriate culture medium. Culture medium may be liquid or semi-solid,e.g. containing agar, methylcellulose, etc. Illustrative examples ofcell culture media include Minimal Essential Media (MEM), Iscove'smodified DMEM, RPMI 1640Clicks, AIM-V, F-12, X-Vivo 15, X-Vivo 20, andOptimizer, with added amino acids, sodium pyruvate, and vitamins, eitherserum-free or supplemented with an appropriate amount of serum (orplasma) or a defined set of hormones, and/or an amount of cytokine(s)sufficient for the growth and expansion of the immune effector cells.

Culture media may be supplemented with one or more factors necessary forproliferation and viability including, but not limited to, growthfactors such as serum (e.g., fetal bovine or human serum at about5%-10%), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21,GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α. Illustrative examples ofother additives for T cell expansion include, but are not limited to,surfactant, piasmanate, pH buffers such as HEPES, and reducing agentssuch as N-acetyl-cysteine and 2-mercaptoethanol, or any other additivessuitable for the growth of cells known to the skilled artisan such asL-glutamine, a thiol, particularly 2-mercaptoethanol, and/orantibiotics, e.g. penicillin and streptomycin. Typically, antibioticsare included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject.

In some embodiments, the population of immune effector cells is obtainedfrom a sample derived from a subject. In some embodiments, a populationof immune effector cells is obtained is obtained from a first subjectand the population of modified immune effector cells produced by themethods described herein is administered to a second, different subject.In some embodiments, a population of immune effector cells is obtainedfrom a subject and the population of modified immune effector cellsproduced by the methods described herein is administered to the samesubject. In some embodiments, the sample is a tissue sample, a fluidsample, a cell sample, a protein sample, or a DNA or RNA sample. In someembodiments, a tissue sample may be derived from any tissue typeincluding, but not limited to skin, hair (including roots), bone marrow,bone, muscle, salivary gland, esophagus, stomach, small intestine (e.g.,tissue from the duodenum, jejunum, or ileum), large intestine, liver,gallbladder, pancreas, lung, kidney, bladder, uterus, ovary, vagina,placenta, testes, thyroid, adrenal gland, cardiac tissue, thymus,spleen, lymph node, spinal cord, brain, eye, ear, tongue, cartilage,white adipose tissue, or brown adipose tissue. In some embodiments, atissue sample may be derived from a cancerous, pre-cancerous, ornon-cancerous tumor. In some embodiments, a fluid sample comprisesbuccal swabs, blood, plasma, oral mucous, vaginal mucous, peripheralblood, cord blood, saliva, semen, urine, ascites fluid, pleural fluid,spinal fluid, pulmonary lavage, tears, sweat, semen, seminal fluid,seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid),excreta, cerebrospinal fluid, lymph, cell culture media comprising oneor more populations of cells, buffered solutions comprising one or morepopulations of cells, and the like.

In some embodiments, the sample is processed to enrich or isolate apopulation of immune effector cells from the remainder of the sample. Incertain embodiments, the sample is a peripheral blood sample which isthen subject to leukapheresis to separate the red blood cells andplatelets and to isolate lymphocytes. In some embodiments, the sample isa leukopak from which immune effector cells can be isolated or enriched.In some embodiments, the sample is a tumor sample that is furtherprocessed to isolate lymphocytes present in the tumor (i.e., byfragmentation and enzymatic digestion of the tumor to obtain a cellsuspension of tumor infiltrating lymphocytes).

In some embodiments, a method for manufacturing modified immune effectorcells contemplated herein comprises activation and/or expansion of apopulation of immune effector cells, as described, for example, in U.S.Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358;6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566;7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. PatentApplication Publication No. 20060121005.

In various embodiments, a method for manufacturing modified immuneeffector cells contemplated herein comprises activating a population ofcells comprising immune effector cells. In particular embodiments, theimmune effector cells are T cells. T cell activation can be accomplishedby providing a primary stimulation signal (e.g., through the T cellTCR/CD3 complex or via stimulation of the CD2 surface protein) and byproviding a secondary co-stimulation signal through an accessorymolecule.

In some embodiments, the TCR/CD3 complex may be stimulated by contactingthe T cell with a suitable CD3 binding agent, e.g., a CD3 ligand or ananti-CD3 monoclonal antibody. Illustrative examples of CD3 antibodiesinclude, but are not limited to, OKT3, G19-4, BC3, CRIS-7 and 64.1. Insome embodiments, a CD2 binding agent may be used to provide a primarystimulation signal to the T cells. Illustrative examples of CD2 bindingagents include, but are not limited to, CD2 ligands and anti-CD2antibodies, e.g., the T11.3 antibody in combination with the T11.1 orT11.2 antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6antibody (which recognizes the same epitope as TI 1.1) in combinationwith the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol.137:1097-1100).

In addition to the stimulatory signal provided through the TCR/CD3complex or CD2, induction of T cell responses typically requires asecond, costimulatory signal provided by a ligand that specificallybinds a costimulatory molecule on a T cell, thereby providing acostimulatory signal which, in addition to the primary signal providedby, for instance, binding of a TCR/CD3 complex, mediates a desired Tcell response. Suitable costimulatory ligands include, but are notlimited to, CD7, B7-1 (CD80), B7-2 (CD86), 4-1BBL, OX40L, induciblecostimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin betareceptor, ILT3, ILT4, an agonist or antibody that binds Toll ligandreceptor, and a ligand that specifically binds with B7-H3.

In some embodiments, a costimulatory ligand comprises an antibody orantigen binding fragment thereof that specifically binds to acostimulatory molecule present on a T cell, including but not limitedto, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3, and aligand that specifically binds with CD83. In particular embodiments, aCD28 binding agent can be used to provide a costimulatory signal.Illustrative examples of CD28 binding agents include but are not limitedto: natural CD28 ligands, e.g., a natural ligand for CD28 (e.g., amember of the B7 family of proteins, such as B7-1 (CD80) and B7-2(CD86); and anti-CD28 monoclonal antibody or fragment thereof capable ofcrosslinking the CD28 molecule, e.g., monoclonal antibodies 9.3, B-T3,XR-CD28, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

In certain embodiments, binding agents that provide stimulatory andcostimulatory signals are localized on the surface of a cell. This canbe accomplished by transfecting or transducing a cell with a nucleicacid encoding the binding agent in a form suitable for its expression onthe cell surface or alternatively by coupling a binding agent to thecell surface. In some embodiments, the costimulatory signal is providedby a costimulatory ligand presented on an antigen presenting cell, suchas an artificial APC (aAPC). Artificial APCs can be made by engineeringK562, U937, 721.221, T2, or C1R cells to stably express and/or secreteof a variety of costimulatory molecules and cytokines to support ex vivogrowth and long-term expansion of genetically modified T cells. In aparticular embodiment, K32 or U32 aAPCs are used to direct the displayof one or more antibody-based stimulatory molecules on the aAPC cellsurface. Populations of T cells can be expanded by aAPCs expressing avariety of costimulatory molecules including, but not limited to, CD137L(4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Exemplary aAPCs areprovided in WO 03/057171 and US2003/0147869, incorporated by referencein their entireties.

In some embodiments, binding agents that provide activating andcostimulatory signals are localized a solid surface (e.g., a bead or aplate). In some embodiments, the binding agents that provide activatingand costimulatory signals are both provided in a soluble form (providedin solution).

In some embodiments, the population of immune effector cells is expandedin culture in one or more expansion phases. “Expansion” refers toculturing the population of immune effector cells for a pre-determinedperiod of time in order to increase the number of immune effector cells.Expansion of immune effector cells may comprise addition of one or moreof the activating factors described above and/or addition of one or moregrowth factors such as a cytokine (e.g., IL-2, IL-15, IL-21, and/orIL-7) to enhance or promote cell proliferation and/or survival. In someembodiments, combinations of IL-2, IL-15, and/or IL-21 can be added tothe cultures during the one or more expansion phases. In someembodiments, the amount of IL-2 added during the one or more expansionphases is less than 6000 U/mL. In some embodiments, the amount of IL-2added during the one or more expansion phases is about 5500 U/mL, about5000 U/mL, about 4500 U/mL, about 4000 U/mL, about 3500 U/mL, about 3000U/mL, about 2500 U/mL, about 2000 U/mL, about 1500 U/mL, about 1000U/mL, or about 500 U/mL. In some embodiments, the amount of IL-2 addedduring the one or more expansion phases is between about 500 U/mL andabout 5500 U/mL. In some embodiments, the population of immune effectorcells may be co-cultured with feeder cells during the expansion process.

In some embodiments, the population of immune effector cells is expandedfor a pre-determined period of time, wherein the pre-determined periodof time is less than about 30 days. In some embodiments, thepre-determined period of time is less than 30 days, less than 25 days,less than 20 days, less than 18 days, less than 15 days, or less than 10days. In some embodiments, the pre-determined period of time is lessthan 4 weeks, less than 3 weeks, less than 2 weeks, or less than 1 week.In some embodiments, the pre-determined period of time is about 7 days,8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16days, 17 days, 18 days, 19 days, 20 days, or 21 days. In someembodiments, the pre-determined period of time is about 5 days to about25 days, about 10 to about 28 days, about 10 to about 25 days, about 10to about 21 days, about 10 to about 20 days, about 10 to about 19 days,about 11 to about 28 days, about 11 to about 25 days, about 11 to about21 days, about 11 to about 20 days, about 11 to about 19 days, about 12to about 28 days, about 12 to about 25 days, about 12 to about 21 days,about 12 to about 20 days, about 12 to about 19 days, about 15 to about28 days, about 15 to about 25 days, about 15 to about 21 days, about 15to about 20 days, or about 15 to about 19 days. In some embodiments, thepre-determined period of time is about 5 days to about 10 days, about 10days to about 15 days, about 15 days to about 20 days, or about 20 daysto about 25 days.

In some embodiments, the population of immune effector cells is expandeduntil the number of cells reaches a pre-determined threshold. Forexample, in some embodiments, the population of immune effector cells isexpanded until the culture comprises at least 5×10⁶, 1×10 1×10⁸, 5×10⁸,1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹, 1×10¹², 5×10¹², 1×10¹³, orat least 5×10¹³ total cells. In some embodiments, the population ofimmune effector cells is expanded until the culture comprises betweenabout 1×10⁹ total cells and about 1×10¹¹ total cells.

In some embodiments, the methods provided herein comprise at least twoexpansion phases. For example, in some embodiments, the population ofimmune effector cells can be expanded after isolation from a sample,allowed to rest, and then expanded again. In some embodiments, theimmune effector cells can be expanded in one set of expansion conditionsfollowed by a second round of expansion in a second, different, set ofexpansion conditions. Methods for ex vivo expansion of immune cells areknown in the art, for example, as described in US Patent ApplicationPublication Nos. 2018-0207201, 20180282694 and 20170152478 and U.S. Pat.Nos. 8,383,099 and 8,034,334, herein incorporated by reference.

At any point during the activation and/or expansion processes, thegene-regulating systems described herein can be introduced to the immuneeffector cells to produce a population of modified immune effectorcells. In some embodiments, the gene-regulating system is introduced tothe population of immune effector cells immediately after enrichmentfrom a sample. In some embodiments, the gene-regulating system isintroduced to the population of immune effector cells before, during, orafter the one or more expansion process. In some embodiments, thegene-regulating system is introduced to the population of immuneeffector cells immediately after enrichment from a sample or harvestfrom a subject, and prior to any expansion rounds. In some embodiments,the gene-regulating system is introduced to the population of immuneeffector cells after a first round of expansion and prior to a secondround of expansion. In some embodiments, the gene-regulating system isintroduced to the population of immune effector cells after a first anda second round of expansion.

In some embodiments, the present disclosure provides methods ofmanufacturing populations of modified immune effector cells comprisingobtaining a population of immune effector cells, introducing agene-regulating system described herein to the population of immuneeffector cells, and expanding the population of immune effector cells inone or more round of expansion. In some aspects of this embodiment, thepopulation of immune effector cells is expanded in a first round ofexpansion prior to the introduction of the gene-regulating system and isexpanded in a second round of expansion after the introduction of thegene-regulating system. In some aspects of this embodiment, thepopulation of immune effector cells is expanded in a first round ofexpansion and a second round of expansion prior to the introduction ofthe gene-regulating system. In some aspects of this embodiment, thegene-regulating system is introduced to the population of immuneeffector cells prior to the first and second rounds of expansion.

In some embodiments, the methods described herein comprise removal of atumor from a subject and processing of the tumor sample to obtain apopulation of tumor infiltrating lymphocytes (e.g., by fragmentation andenzymatic digestion of the tumor to obtain a cell suspension)introducing a gene-regulating system described herein to the populationof immune effector cells, and expanding the population of immuneeffector cells in one or more round of expansion. In some aspects ofthis embodiment, the population of tumor infiltrating lymphocytes isexpanded in a first round of expansion prior to the introduction of thegene-regulating system and is expanded in a second round of expansionafter the introduction of the gene-regulating system. In some aspects ofthis embodiment, the population of tumor infiltrating lymphocytes isexpanded in a first round of expansion and a second round of expansionprior to the introduction of the gene-regulating system. In some aspectsof this embodiment, the gene-regulating system is introduced to thepopulation of tumor infiltrating lymphocytes prior to the first andsecond rounds of expansion.

In some embodiments, the modified immune effector cells produced by themethods described herein may be used immediately. In some embodiments,the manufacturing methods contemplated herein may further comprisecryopreservation of modified immune cells for storage and/or preparationfor use in a subject. As used herein, “cryopreserving,” refers to thepreservation of cells by cooling to sub-zero temperatures, such as(typically) 77 K or −196° C. (the boiling point of liquid nitrogen). Insome embodiments, a method of storing modified immune effector cellscomprises cryopreserving the immune effector cells such that the cellsremain viable upon thawing. When needed, the cryopreserved modifiedimmune effector cells can be thawed, grown and expanded for more suchcells. Cryoprotective agents are often used at sub-zero temperatures toprevent the cells being preserved from damage due to freezing at lowtemperatures or warming to room temperature. Cryopreservative agents andoptimal cooling rates can protect against cell injury. Cryoprotectiveagents which can be used include but are not limited to dimethylsulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395;Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol,polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960; 85: 576), andpolyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). Insome embodiments, the cells are frozen in 10% dimethylsulfoxide (DMSO),50% serum, 40% buffered medium, or some other such solution as iscommonly used in the art to preserve cells at such freezingtemperatures, and thawed in a manner as commonly known in the art forthawing frozen cultured cells.

A. Producing Modified Immune Effector Cells Using CRISPR/Cas Systems

In some embodiments, a method of producing a modified immune effectorcell involves contacting a target DNA sequence with a complex comprisinga gRNA and a Cas polypeptide. As discussed above, a gRNA and Caspolypeptide form a complex, wherein the DNA-binding domain of the gRNAtargets the complex to a target DNA sequence and wherein the Cas protein(or heterologous protein fused to an enzymatically inactive Cas protein)modifies target DNA sequence. In some embodiments, this complex isformed intracellularly after introduction of the gRNA and Cas protein(or polynucleotides encoding the gRNA and Cas proteins) to a cell. Insome embodiments, the nucleic acid encoding the Cas protein is a DNAnucleic acid and is introduced to the cell by transduction. In someembodiments, the Cas9 and gRNA components of a CRISPR/Cas gene editingsystem are encoded by a single polynucleotide molecule. In someembodiments, the polynucleotide encoding the Cas protein and gRNAcomponent are comprised in a viral vector and introduced to the cell byviral transduction. In some embodiments, the Cas9 and gRNA components ofa CRISPR/Cas gene editing system are encoded by different polynucleotidemolecules. In some embodiments, the polynucleotide encoding the Casprotein is comprised in a first viral vector and the polynucleotideencoding the gRNA is comprised in a second viral vector. In some aspectsof this embodiment, the first viral vector is introduced to a cell priorto the second viral vector. In some aspects of this embodiment, thesecond viral vector is introduced to a cell prior to the first viralvector. In such embodiments, integration of the vectors results insustained expression of the Cas9 and gRNA components. However, sustainedexpression of Cas9 may lead to increased off-target mutations andcutting in some cell types. Therefore, in some embodiments, an mRNAnucleic acid sequence encoding the Cas protein may be introduced to thepopulation of cells by transfection. In such embodiments, the expressionof Cas9 will decrease over time, and may reduce the number of off targetmutations or cutting sites. In some embodiments, the gRNA and Casprotein are introduced separately by electroporation.

In some embodiments, this complex is formed in a cell-free system bymixing the gRNA molecules and Cas proteins together and incubating for aperiod of time sufficient to allow complex formation. This pre-formedcomplex, comprising the gRNA and Cas protein and referred to herein as aCRISPR-ribonucleoprotein (CRISPR-RNP) can then be introduced to a cellin order to modify a target DNA sequence. In some embodiments, theCRISPR-RNP is introduced to the cell by electroporation.

In any of the above described embodiments for producing a modifiedimmune effector cell using the CRISPR/Cas system, the system maycomprise one or more gRNAs targeting a single endogenous target gene,for example to produce a single-edited modified immune effector cell.Alternatively, in any of the above described embodiments for producing amodified immune effect cell using the CRISPR/Cas system, the system maycomprise two or more gRNAs targeting two or more endogenous targetgenes, for example to produce a dual-edited modified immune effectorcell.

B. Producing Modified Immune Effector Cells Using shRNA Systems

In some embodiments, a method of producing a modified immune effectorcell introducing into the cell one or more DNA polynucleotides encodingone or more shRNA molecules with sequence complementary to the mRNAtranscript of a target gene. The immune effector cell can be modified toproduce the shRNA by introducing specific DNA sequences into the cellnucleus via a small gene cassette. Both retroviruses and lentivirusescan be used to introduce shRNA-encoding DNAs into immune effector cells.The introduced DNA can either become part of the cell's own DNA orpersist in the nucleus, and instructs the cell machinery to produceshRNAs. shRNAs may be processed by Dicer or AGO2-mediated sliceractivity inside the cell to induce RNAi mediated gene knockdown.

VI. Compositions and Kits

The term “composition” as used herein refers to a formulation of agene-regulating system or a modified immune effector cell describedherein that is capable of being administered or delivered to a subjector cell. Typically, formulations include all physiologically acceptablecompositions including derivatives and/or prodrugs, solvates,stereoisomers, racemates, or tautomers thereof with any physiologicallyacceptable carriers, diluents, and/or excipients. A “therapeuticcomposition” or “pharmaceutical composition” (used interchangeablyherein) is a composition of a gene-regulating system or a modifiedimmune effector cell capable of being administered to a subject for thetreatment of a particular disease or disorder or contacted with a cellfor modification of one or more endogenous target genes.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier whichhas been approved by the United States Food and Drug Administration asbeing acceptable for use in humans and/or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations. Except insofar as anyconventional media and/or agent is incompatible with the agents of thepresent disclosure, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts. Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

In some embodiments, the present disclosure provides kits for carryingout a method described herein. In some embodiments, a kit can include:

(a) one or more nucleic acid molecules capable of reducing theexpression or modifying the function of a gene product encoded by one ormore endogenous target genes;

(b) one or more polynucleotides encoding a nucleic acid molecule that iscapable of reducing the expression or modifying the function of a geneproduct encoded by one or more endogenous target genes;

(c) one or more proteins capable of reducing the expression or modifyingthe function of a gene product encoded by one or more endogenous targetgenes;

(d) one or more polynucleotides encoding a modifying protein that iscapable of reducing the expression or modifying the function of a geneproduct encoded by one or more endogenous target genes;

(e) one or more gRNAs capable of binding to a target DNA sequence in anendogenous gene;

(f) one or more polynucleotides encoding one or more gRNAs capable ofbinding to a target DNA sequence in an endogenous gene;

(g) one or more site-directed modifying polypeptides capable ofinteracting with a gRNA and modifying a target DNA sequence in anendogenous gene;

(h) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gRNA and modifying a targetDNA sequence in an endogenous gene;

(i) one or more guide DNAs (gDNAs) capable of binding to a target DNAsequence in an endogenous gene;

(j) one or more polynucleotides encoding one or more gDNAs capable ofbinding to a target DNA sequence in an endogenous gene;

(k) one or more site-directed modifying polypeptides capable ofinteracting with a gDNA and modifying a target DNA sequence in anendogenous gene;

(l) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gDNA and modifying a targetDNA sequence in an endogenous gene;

(m) one or more gRNAs capable of binding to a target mRNA sequenceencoded by an endogenous gene;

(n) one or more polynucleotides encoding one or more gRNAs capable ofbinding to a target mRNA sequence encoded by an endogenous gene;

(o) one or more site-directed modifying polypeptides capable ofinteracting with a gRNA and modifying a target mRNA sequence encoded byan endogenous gene;

(p) one or more polynucleotides encoding a site-directed modifyingpolypeptide capable of interacting with a gRNA and modifying a targetmRNA sequence encoded by an endogenous gene;

(q) a modified immune effector cell described herein; or

(r) any combination of the above.

In some embodiments, the kits described herein further comprise one ormore immune checkpoint inhibitors. Several immune checkpoint inhibitorsare known in the art and have received FDA approval for the treatment ofone or more cancers. For example, FDA-approved PD-L1 inhibitors includeAtezolizumab (Tecentriq®, Genentech), Avelumab (Bavencio®, Pfizer), andDurvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-1 inhibitors includePembrolizumab (Keytruda®, Merck) and Nivolumab (Opdivo®, Bristol-MyersSquibb); and FDA-approved CTLA4 inhibitors include Ipilimumab (Yervoy®,Bristol-Myers Squibb). Additional inhibitory immune checkpoint moleculesthat may be the target of future therapeutics include A2AR, B7-H3,B7-H4, BTLA, IDO, LAG3 (e.g., BMS-986016, under development by BSM), KIR(e.g., Lirilumab, under development by BSM), TIM3, TIGIT, and VISTA.

In some embodiments, the kits described herein comprise one or morecomponents of a gene-regulating system (or one or more polynucleotidesencoding the one or more components) and one or more immune checkpointinhibitors known in the art (e.g., a PD1 inhibitor, a CTLA4 inhibitor, aPDL1 inhibitor, etc.). In some embodiments, the kits described hereincomprise one or more components of a gene-regulating system (or one ormore polynucleotides encoding the one or more components) and ananti-PD1 antibody (e.g., Pembrolizumab or Nivolumab). In someembodiments, the kits described herein comprise a modified immuneeffector cell described herein (or population thereof) and one or moreimmune checkpoint inhibitors known in the art (e.g., a PD1 inhibitor, aCTLA4 inhibitor, a PDL1 inhibitor, etc.). In some embodiments, the kitsdescribed herein comprise a modified immune effector cell describedherein (or population thereof) and an anti-PD1 antibody (e.g.,Pembrolizumab or Nivolumab).

In some embodiments, the kit comprises one or more components of agene-regulating system (or one or more polynucleotides encoding the oneor more components) and a reagent for reconstituting and/or diluting thecomponents. In some embodiments, a kit comprising one or more componentsof a gene-regulating system (or one or more polynucleotides encoding theone or more components) and further comprises one or more additionalreagents, where such additional reagents can be selected from: a bufferfor introducing the gene-regulating system into a cell; a wash buffer; acontrol reagent; a control expression vector or RNA polynucleotide; areagent for in vitro production of the gene-regulating system from DNA,and the like. Components of a kit can be in separate containers or canbe combined in a single container.

In addition to above-mentioned components, in some embodiments a kitfurther comprises instructions for using the components of the kit topractice the methods of the present disclosure. The instructions forpracticing the methods are generally recorded on a suitable recordingmedium. For example, the instructions may be printed on a substrate,such as paper or plastic, etc. As such, the instructions may be presentin the kits as a package insert or in the labeling of the container ofthe kit or components thereof (i.e., associated with the packaging orsub-packaging). In other embodiments, the instructions are present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In yet otherembodiments, the actual instructions are not present in the kit, butmeans for obtaining the instructions from a remote source, e.g. via theinternet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions is recorded on a suitablesubstrate.

VII. Therapeutic Methods and Applications

In some embodiments, the modified immune effector cells andgene-regulating systems described herein may be used in a variety oftherapeutic applications. For example, in some embodiments the modifiedimmune effector cells and/or gene-regulating systems described hereinmay be administered to a subject for purposes such as gene therapy, e.g.to treat a disease, for use as an antiviral, for use as ananti-pathogenic, for use as an anti-cancer therapeutic, or forbiological research.

In some embodiments, the subject may be a neonate, a juvenile, or anadult. Of particular interest are mammalian subjects. Mammalian speciesthat may be treated with the present methods include canines andfelines; equines; bovines; ovines; etc. and primates, particularlyhumans. Animal models, particularly small mammals (e.g. mice, rats,guinea pigs, hamsters, rabbits, etc.) may be used for experimentalinvestigations.

Administration of the modified immune effector cells described herein,populations thereof, and compositions thereof can occur by injection,irrigation, inhalation, consumption, electro-osmosis, hemodialysis,iontophoresis, and other methods known in the art. In some embodiments,administration route is local or systemic. In some embodimentsadministration route is intraarterial, intracranial, intradermal,intraduodenal, intrammamary, intrameningeal, intraperitoneal,intrathecal, intratumoral, intravenous, intravitreal, ophthalmic,parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, orintrauterine.

In some embodiments, the administration route is by infusion (e.g.,continuous or bolus). Examples of methods for local administration, thatis, delivery to the site of injury or disease, include through an Ommayareservoir, e.g. for intrathecal delivery (See e.g., U.S. Pat. Nos.5,222,982 and 5,385,582, incorporated herein by reference); by bolusinjection, e.g. by a syringe, e.g. into a joint; by continuous infusion,e.g. by cannulation, such as with convection (See e.g., US PatentApplication Publication No. 2007-0254842, incorporated herein byreference); or by implanting a device upon which the cells have beenreversibly affixed (see e.g. US Patent Application Publication Nos.2008-0081064 and 2009-0196903, incorporated herein by reference). Insome embodiments, the administration route is by topical administrationor direct injection. In some embodiments, the modified immune effectorcells described herein may be provided to the subject alone or with asuitable substrate or matrix, e.g. to support their growth and/ororganization in the tissue to which they are being transplanted.

In some embodiments, at least 1×10³ cells are administered to a subject.In some embodiments, at least 5×10³ cells, 1×10⁴ cells, 5×10⁴ cells,1×10⁵ cells, 5×10⁵ cells, 1×10⁶ 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹,1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹, 1×10¹², 5×10¹², or more cells areadministered to a subject. In some embodiments, between about 1×10⁷ andabout 1×10¹² cells are administered to a subject. In some embodiments,between about 1×10⁸ and about 1×10¹² cells are administered to asubject. In some embodiments, between about 1×10⁹ and about 1×10¹² cellsare administered to a subject. In some embodiments, between about 1×10¹⁰and about 1×10¹² cells are administered to a subject. In someembodiments, between about 1×10¹¹ and about 1×10¹² cells areadministered to a subject. In some embodiments, between about 1×10⁷ andabout 1×10¹¹ cells are administered to a subject. In some embodiments,between about 1×10⁷ and about 1×10¹⁰ cells are administered to asubject. In some embodiments, between about 1×10⁷ and about 1×10⁹ cellsare administered to a subject. In some embodiments, between about 1×10⁷and about 1×10⁸ cells are administered to a subject. The number ofadministrations of treatment to a subject may vary. In some embodiments,introducing the modified immune effector cells into the subject may be aone-time event. In some embodiments, such treatment may require anon-going series of repeated treatments. In some embodiments, multipleadministrations of the modified immune effector cells may be requiredbefore an effect is observed. The exact protocols depend upon thedisease or condition, the stage of the disease and parameters of theindividual subject being treated.

In some embodiments, the gene-regulating systems described herein areemployed to modify cellular DNA or RNA in vivo, such as for gene therapyor for biological research. In such embodiments, a gene-regulatingsystem may be administered directly to the subject, such as by themethods described supra. In some embodiments, the gene-regulatingsystems described herein are employed for the ex vivo or in vitromodification of a population of immune effector cells. In suchembodiments, the gene-regulating systems described herein areadministered to a sample comprising immune effector cells.

In some embodiments, the modified immune effector cells described hereinare administered to a subject. In some embodiments, the modified immuneeffector cells described herein administered to a subject are autologousimmune effector cells. The term “autologous” in this context refers tocells that have been derived from the same subject to which they areadministered. For example, immune effector cells may be obtained from asubject, modified ex vivo according to the methods described herein, andthen administered to the same subject in order to treat a disease. Insuch embodiments, the cells administered to the subject are autologousimmune effector cells. In some embodiments, the modified immune effectorcells, or compositions thereof, administered to a subject are allogenicimmune effector cells. The term “allogenic” in this context refers tocells that have been derived from one subject and are administered toanother subject. For example, immune effector cells may be obtained froma first subject, modified ex vivo according to the methods describedherein and then administered to a second subject in order to treat adisease. In such embodiments, the cells administered to the subject areallogenic immune effector cells.

In some embodiments, the modified immune effector cells described hereinare administered to a subject in order to treat a disease. In someembodiments, treatment comprises delivering an effective amount of apopulation of cells (e.g., a population of modified immune effectorcells) or composition thereof to a subject in need thereof. In someembodiments, treating refers to the treatment of a disease in a mammal,e.g., in a human, including (a) inhibiting the disease, i.e., arrestingdisease development or preventing disease progression; (b) relieving thedisease, i.e., causing regression of the disease state or relieving oneor more symptoms of the disease; and (c) curing the disease, i.e.,remission of one or more disease symptoms. In some embodiments,treatment may refer to a short-term (e.g., temporary and/or acute)and/or a long-term (e.g., sustained) reduction in one or more diseasesymptoms. In some embodiments, treatment results in an improvement orremediation of the symptoms of the disease. The improvement is anobservable or measurable improvement, or may be an improvement in thegeneral feeling of well-being of the subject.

The effective amount of a modified immune effector cell administered toa particular subject will depend on a variety of factors, several ofwhich will differ from patient to patient including the disorder beingtreated and the severity of the disorder; activity of the specificagent(s) employed; the age, body weight, general health, sex and diet ofthe patient; the timing of administration, route of administration; theduration of the treatment; drugs used in combination; the judgment ofthe prescribing physician; and like factors known in the medical arts.

In some embodiments, the effective amount of a modified immune effectorcell may be the number of cells required to result in at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more folddecrease in tumor mass or volume, decrease in the number of tumor cells,or decrease in the number of metastases. In some embodiments, theeffective amount of a modified immune effector cell may be the number ofcells required to achieve an increase in life expectancy, an increase inprogression-free or disease-free survival, or amelioration of variousphysiological symptoms associated with the disease being treated. Insome embodiments, an effective amount of modified immune effector cellswill be at least 1×10³ cells, for example 5×10³ cells, 1×10⁴ cells,5×10⁴ cells, 1×10⁵ cells, 5×10⁵ cells, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, 1×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹,5×10¹¹, 1×10¹², 5×10¹², or more cells.

In some embodiments, the modified immune effector cells andgene-regulating systems described herein may be used in the treatment ofa cell-proliferative disorder, such as a cancer. Cancers that may betreated using the compositions and methods disclosed herein includecancers of the blood and solid tumors. For example, cancers that may betreated using the compositions and methods disclosed herein include, butare not limited to, adenoma, carcinoma, sarcoma, leukemia or lymphoma.In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), Bcell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL),diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma(DLBCL), Hodgkin's lymphoma, multiple myeloma, renal cell carcinoma(RCC), neuroblastoma, colorectal cancer, bladder cancer, breast cancer,colorectal cancer, ovarian cancer, melanoma, sarcoma, prostate cancer,lung cancer, esophageal cancer, hepatocellular carcinoma, pancreaticcancer, astrocytoma, mesothelioma, head and neck cancer, andmedulloblastoma, and liver cancer. In some embodiments, the cancer isselected from a melanoma, head and neck cancer, bladder cancer, lungcancer, cervical cancer, pancreatic cancer, breast cancer, andcolorectal cancer. In some embodiments, the cancer is insensitive, orresistant, to treatment with a PD1 inhibitor. In some embodiments, thecancer is insensitive, or resistant to treatment with a PD1 inhibitorand is selected from a melanoma, head and neck cancer, bladder cancer,lung cancer, cervical cancer, pancreatic cancer, breast cancer, andcolorectal cancer.

As described above, several immune checkpoint inhibitors are currentlyapproved for use in a variety of oncologic indications (e.g., CTLA4inhibitors, PD1 inhibitors, PDL1 inhibitors, etc.). In some embodiments,administration of a modified immune effector cell comprising reducedexpression and/or function of an endogenous target gene described hereinresults in an enhanced therapeutic effect (e.g., a more significantreduction in tumor growth, an increase in tumor infiltration bylymphocytes, an increase in the length of progression free survival,etc.) than is observed after treatment with an immune checkpointinhibitor.

Further, some oncologic indications are non-responsive (i.e., areinsensitive) to treatment with immune checkpoint inhibitors. Furtherstill, some oncologic indications that are initially responsive (i.e.,sensitive) to treatment with immune checkpoint inhibitors develop aninhibitor-resistant phenotype during the course of treatment. Therefore,in some embodiments, the modified immune effector cells describedherein, or compositions thereof, are administered to treat a cancer thatis resistant (or partially resistant) or insensitive (or partiallyinsensitive) to treatment with one or more immune checkpoint inhibitors.In some embodiments, administration of the modified immune effectorcells or compositions thereof to a subject suffering from a cancer thatis resistant (or partially resistant) or insensitive (or partiallyinsensitive) to treatment with one or more immune checkpoint inhibitorsresults in treatment of the cancer (e.g., reduction in tumor growth, anincrease in the length of progression free survival, etc.). In someembodiments, the cancer is resistant (or partially resistant) orinsensitive (or partially insensitive) to treatment with a PD1inhibitor.

In some embodiments, the modified immune effector cells or compositionsthereof are administered in combination with an immune checkpointinhibitor. In some embodiments, administration of the modified immuneeffector cells in combination with the immune checkpoint inhibitorresults in an enhanced therapeutic effect in a cancer that is resistant,refractory, or insensitive to treatment by an immune checkpointinhibitor than is observed by treatment with either the modified immuneeffector cells or the immune checkpoint inhibitor alone. In someembodiments, administration of the modified immune effector cells incombination with the immune checkpoint inhibitor results in an enhancedtherapeutic effect in a cancer that is partially resistant, partiallyrefractory, or partially insensitive to treatment by an immunecheckpoint inhibitor than is observed by treatment with either themodified immune effector cells or the immune checkpoint inhibitor alone.In some embodiments, the cancer is resistant (or partially resistant),refractory (or partially refractory), or insensitive (or partiallyinsensitive) to treatment with a PD1 inhibitor.

In some embodiments, administration of a modified immune effector celldescribed herein or composition thereof in combination with an anti-PD1antibody results in an enhanced therapeutic effect in a cancer that isresistant or insensitive to treatment by the anti-PD1 antibody alone. Insome embodiments, administration of a modified immune effector celldescribed herein or composition thereof in combination with an anti-PD1antibody results in an enhanced therapeutic effect in a cancer that ispartially resistant or partially insensitive to treatment by theanti-PD1 antibody alone.

Cancers that demonstrate resistance or sensitivity to immune checkpointinhibition are known in the art and can be tested in a variety of invivo and in vitro models. For example, some melanomas are sensitive totreatment with an immune checkpoint inhibitor such as an anti-PD1antibody and can be modeled in an in vivo B16-Ova tumor model (SeeExamples 6, 7, 16, and 19). Further, some colorectal cancers are knownto be resistant to treatment with an immune checkpoint inhibitor such asan anti-PD1 antibody and can be modeled in a PMEL/MC38-gp100 model (SeeExamples 8 and 17). Further still, some lymphomas are known to beinsensitive to treatment with an immune checkpoint inhibitor such as ananti-PD1 antibody and can be modeled in a various models by adoptivetransfer or subcutaneous administration of lymphoma cell lines, such asRaji cells (See Examples 12, 14, 15, 21, and 22).

In some embodiments, the modified immune effector cells andgene-regulating systems described herein may be used in the treatment ofa viral infection. In some embodiments, the virus is selected from oneof adenoviruses, herpesviruses (including, for example, herpes simplexvirus and Epstein Barr virus, and herpes zoster virus), poxviruses,papovaviruses, hepatitis viruses, (including, for example, hepatitis Bvirus and hepatitis C virus), papilloma viruses, orthomyxoviruses(including, for example, influenza A, influenza B, and influenza C),paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses,flaviviruses, bunyaviridae, rhabdoviruses, rotavirus, respiratorysyncitial virus, human immunodeficiency virus, or retroviruses.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not be taken as, an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world.

EXAMPLES Example 1: Materials and Methods

The experiments described herein utilize the CRISPR/Cas9 system tomodulate expression of one or more endogenous target genes in differentT cell populations.

I. Materials

gRNAs: Unless otherwise indicated, all experiments use single-moleculegRNAs (sgRNAs). Dual gRNA molecules were used as indicated and wereformed by duplexing 200 μM tracrRNA (IDT Cat #1072534) with 200 μM oftarget-specific crRNA (IDT) in nuclease free duplex buffer (IDT Cat#11-01-03-01) for 5 min at 95° C., to form 100 μM of tracrRNA:crRNAduplex, where the tracrRNA and crRNA are present at a 1:1 ratio.Targeting sequences of the gRNAs used in the following experiments areprovided in Table 10 below.

TABLE 10 Experiment gRNA targeting-domain encoding sequences SEQTarget Gene Guide ID Sequence ID Pdcd1 - murine Nm.Pdcd1CGGAGGATCTTATGCTGAAC 778 Lag3 - murine Nm.Lag3 GCCAAGTGGACTCCTCCTGG 602Cblb - murine Nm.Cblb CCTTATCTTCAGTCACATGC 502 CBLB - human Nm.CBLBTAAACTTACCTGAAACAGCC 521 BCOR - human Nm.BCOR GTGCAGACTGGAGAATACAG 715ZC3h12a - murine Nm.Zc3h12a TTCCCTCCTCTGCCAGCCAT 1505 ZC3H12A - humanNm.ZC3H12A_1 GGAGTGGAAGCGCTTCATCG 1434 Nr4a3 - murine Nm.Nr4a3GGCAGCGTTGTCGCCGCACA 1544 Map4k1 - murine Nm.Map4k1 TTGCCGGCACGCCAACATCG1515 OR1A2 - human Nh.OR1A2_1 AGATGATGTCAACCAAGGAG 1574 Olfr421 - mouseNm.Olfr421_1 GGAAGAAGTACATCTGCAAG 1574

Cas9: Cas9 was expressed in target cells by introduction of either Cas9mRNA or a Cas9 protein. Unless otherwise indicated, Cas9-encoding mRNAcomprising a nuclear localization sequence (Cas9-NLS mRNA) derived fromS. pyogenes (Trilink L-7206) or Cas9 protein derived from S. pyogenes(IDT Cat #1074182) was used in the following experiments.

RNPs: Human gRNA-Cas9 ribonucleoproteins (RNPs) were formed by combining1.2 μL of 100 μM tracrRNA:crRNA duplex with 1 μL of 20 μM Cas9 proteinand 0.8 μL of PBS. Mixtures were incubated at RT for 20 minutes to formthe RNP complexes. Murine RNPs were formed by combining 1 Volume of 44μM tracrRNA:crRNA duplex with 1 Volume of 36 μM Cas9. Mixtures wereincubated at RT for 20 minutes to form the RNP complexes.

Mice: Wild type CD8⁺ T cells were derived from C57BL/6J mice (TheJackson Laboratory, Bar Harbor Me.). Ovalbumin (Ova)-specific CD8⁺ Tcells were derived from OT1 mice (C57BL/6-Tg(TcraTcrb) 1100Mjb/J;Jackson Laboratory). OT1 mice comprise a transgenic TCR that recognizesresidues 257-264 of the ovalbumin (Ova) protein. gp100-specific CD8+ Tcells were derived from PMEL mice (B6.Cg-Thy1<a>/CyTg(TcraTcrb) 8Rest/J;The Jackson Laboratory, Bar Harbor Me. Cat #005023). Mice constitutivelyexpressing the Cas9 protein were obtain from Jackson labs(B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J; The JacksonLaboratory, Bar Harbor Me. Strain #026179), TCR-transgenic miceconstitutively expressing Cas9 were obtained by breeding of OT1 and PMELmice with Cas9 mice.

CAR Expression Constructs: CARs specific for human CD19, Her2/Erbb2, andEGFR proteins were generated. Briefly, the 22 amino acid signal peptideof the human granulocyte-macrophage colony stimulating factor receptorsubunit alpha (GMSCF-Rα) was fused to an antigen-specific scFv domainspecifically binding to one of CD19, Her2/Erbb2, or EGFR. The human CD8astalk was used as a transmembrane domain. The intracellular signalingdomains of the CD3 chain were fused to the cytoplasmic end of the CD8astalk. For anti-CD19 CARs, the scFv was derived from the anti-human CD19clone FMC63. To create a CAR specific for human HER2/ERBB2, theanti-human HER2 scFv derived from trastuzumab was used. Similarly, togenerate a CAR specific for EGFR, the anti-EGFR scFv derived fromcetuximab was used. A summary of exemplary CAR constructs is shown belowand amino acid sequences of the full length CAR constructs are providedin SEQ ID NOs: 26, 28, and 30, and nucleic acid sequences of the fulllength CAR constructs are provided in SEQ ID NOs: 27, 29, and 31.

TABLE 11 Exemplary CAR constructs Ag-binding Intracellular TransmembraneAA NA CAR Ref ID Target domain Domain Domain SEQ ID SEQ ID KSQCAR017human Cetuximab CD3 zeta CD8a hinge 26 27 EGFR H225 scFv KSQCAR1909human FMC63 scFv CD3 zeta CD8a hinge 28 28 CD19 KSQCAR010 humanHerceptin CD3 zeta CD8a hinge 30 31 HER2 scFv

Engineered TCRs Expression Constructs: Recombinant TCRs specific forNY-ESO1, MART-1, and WT-1 were generated. Paired TCR-α:TCR-β variableregion protein sequences encoding the 1G4 TCR specific for the NY-ESO-1peptide SLLMWITQC (SEQ ID NO: 2), the DMF4 and DMF5 TCRs specific forthe MART-1 peptide AAGIGILTV (SEQ ID NO: 3), and the DLT andhigh-affinity DLT TCRs specific for the WT-1 peptide, each presented byHLA-A*02:01, were identified from the literature (Robbins et al, Journalof Immunology 2008 180:6116-6131). TCRα chains were composed of V and Jgene segments and CDR3α sequences and TCRβ chains were composed of V, D,and J gene segment and CDR3-β sequences. The native TRAC (SEQ ID NO: 22)and TRBC (SEQ ID NOs: 24) protein sequences were fused to the C-terminalends of the α and β chain variable regions, respectively, to produce1G4-TCR α/βchains (SEQ ID NOs: 11 and 12, respectively), 95:LY 1G4-TCRα/βchains (SEQ ID NOs: 14 and 13, respectively), DLT-TCR α/βchains (SEQID NOs: 5 and 4, respectively), high-affinity DLT-TCR α/βchains (SEQ IDNOs: 8 and 7, respectively), DMF4-TCR α/βchains (SEQ ID NOs: 17 and 16,respectively), and DMF5-TCR α/βchains (SEQ ID NOs: 20 and 19,respectively).

Codon-optimized DNA sequences encoding the engineered TCRα and TCRβchain proteins were generated where the P2A sequence (SEQ ID NO: 1) wasinserted between the DNA sequences encoding the TCRβ and the TCRα chain,such that expression of both TCR chains was driven off of a singlepromoter in a stoichiometric fashion. The expression cassettes encodingthe engineered TCR chains therefore comprised the following format:TCRβ−P2A−TCRα. Final protein sequences for each TCR construct areprovided in SEQ ID NO: 12 (1G4), SEQ ID NO: 15 (95:LY 1G4), SEQ ID NO: 6(DLT), SEQ ID NO: 9 (high-affinity DLT), SEQ ID NO: 18 (DMF4), and SEQID NO: 21 (DMF5).

Lentiviral Expression of CAR and TCR Constructs: The CAR and engineeredTCR expression constructs described above were then inserted into aplasmid comprising an SFFV promoter driving expression of the engineeredreceptor, a T2A sequence, and a puromycin resistance cassette. Unlessotherwise indicated, these plasmids further comprised a human or amurine (depending on the species the T cells were derived from) U6promoter driving expression of one or more sgRNAs. Lentivirus constructscomprising an engineered TCR expression construct may further comprisean sgRNA targeting the endogenous TRAC gene, which encodes the constantregion of the α chain of the T cell receptor. Lentiviruses encoding theengineered receptors described above were generated as follows. Briefly,289×10⁶ of LentiX-293T cells were plated out in a 5-layer CellSTACK 24hours prior to transfection. Serum-free OptiMEM and TransIT-293 werecombined and incubated for 5 minutes before combining helper plasmids(58 μg VSVG and 115 μg PAX2-Gag-Pol) with 231 μg of an engineeredreceptor- and sgRNA-expressing plasmid described above. After 20minutes, this mixture was added to the LentiX-293T cells with freshmedia. Media was replaced 18 hours after transfection and viralsupernatants were collected 48 hours post-transfection. Supernatantswere treated with Benzonase® nuclease and passed through a 0.45 μmfilter to isolate the viral particles. Virus particles were thenconcentrated by Tangential Flow Filtration (TFF), aliquoted, tittered,and stored at −80° C.

Retroviral sgRNA expression constructs and production: sgRNA sequenceswere inserted into a plasmid downstream of a murine U6 promoter. HumanCD2 was inserted under the UbiC promoter downstream of the sgRNA as aselectable marker. When rescue experiments were conducted, plasmidsincluded the above elements as well as a codon optimized, gRNA-resistantcDNA encoding wild-type murine Zc3h12a. Retroviruses were generated asfollows. Phoenix-GP cells (ATCC® CRL-3215™) were used as producer cells.When 80% confluent, producer cells were transfected with pCL-EcoRetrovirus Packaging Vector and the plasmid encoding the sgRNA andsurface selection marker described above using TransIT®-293 TransfectionReagent (Mirusbio, Catalog #2706). 18 hours after transfection, mediawas changed for complete T cell media (RPMI+10% heat inactivated FBS, 20mM HEPES, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 50 μMBeta-Mercaptoethanol). Viral supernatants were harvested every 12 hoursfor a total of 3 harvests, spun and frozen at −80° C.

II. Methods

Human T cell Isolation and Activation: Total human PBMCs were isolatedfrom fresh leukopacks by Ficoll gradient centrifugation. CD8+ T-cellswere then purified from total PBMCs using a CD8+ T-cell isolation kit(Stemcell Technologies Cat #17953). For T cell activation, CD8+ T cellswere plated at 2×106 cells/mL in X-VIVO 15 T Cell Expansion Medium(Lonza, Cat #04-418Q) in a T175 flask, with 6.25 μL/mL of ImmunoCultT-cell activators (anti-CD3/CD28/CD2, StemCell Technologies, VancouverBC, Canada) and 10 ng/mL human IL2. T-cells were activated for 18 hoursprior to transduction with lentiviral constructs.

Human TIL Isolation and Activation: Tumor infiltrating lymphocytes canalso be modified by the methods described herein. In such cases, tumorsare surgically resected from human patients and diced with scalpelblades into 1 mm 3 pieces, with a single piece of tumor placed into eachwell of a 24 plate. 2 mL of complete TIL media (RPMI+10% heatinactivated human male AB serum, 1 mM pyruvate, 20 μg/mL gentamycin, 1×glutamax) supplemented with 6000 U/mL of recombinant human IL-2 is addedto each well of isolated TILs. 1 mL of media is removed from the welland replaced with fresh media and IL-2 up to 3 times a week as needed.As wells reach confluence, they are split 1:1 in new media+IL-2. After4-5 weeks of culture, the cells are harvested for rapid expansion.

TIL Rapid Expansion: TILs are rapidly expanded by activating 500,000TILs with 26×106 allogeneic, irradiated (5000cGy) PBMC feeder cells in20 mL TIL media+20 mL of Aim-V media (Invitrogen)+30 ng/mL OKT3 mAb. 48hours later (Day 2), 6000 U/mL IL-2 is added to the cultures. On day 5,20 mL of media is removed, and 20 mL fresh media (+30 ng/ml OKT3) isadded. On Day 7, cells are counted, and reseeded at 60×106 cells/L inG-Rex6M well plates (Wilson Wolf, Cat #80660M) or G-Rex100M (WilsonWolf, Cat #81100S), depending on the number of cells available. 6000U/mL fresh IL-2 is added on Day 9 and 3000 U/mL fresh IL-2 is added onDay 12. TILs are harvested on Day 14. Expanded cells are thenslow-frozen in Cryostor CS-10 (Stemcell Technologies Cat #07930) usingCoolcell Freezing containers (Corning) and stored long term in liquidnitrogen.

Murine T cell Isolation and Activation: Spleens from WT or transgenicmice were harvested and reduced to a single cell suspension using theGentleMACS system, according to the manufacturer's recommendations.Purified CD8⁺ T cells were obtained using the EasySep Mouse CD8⁺ T CellIsolation Kit (StemCell Catalog #19853). CD8 T-cells were cultured at1×106 cells/mL in complete T cell media (RPMI+10% heat inactivated FBS,20 mM HEPES, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 50 μMBeta-Mercaptoethanol) supplemented with 2 ng/mL of Recombinant MouseIL-2 (Biolegend Catalog #575406) and activated with anti-CD3/anti-CD28beads (Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion andActivation Cat #11456D).

Lentiviral transduction of human T cells: T-cells activated 18 hoursprior were seeded at 5×10⁶ cells per well in a 6 well plate, in 1.5 mLvolume of X-VIVO 15 media, 10 ng/mL human IL-2 and 12.5 μL ImmunocultHuman CD3/CD28/CD2 T-cell Activator. Lentivirus expressing theengineered receptors was added at an MOI capable of infecting 80% of allcells. 25 μL of Retronectin (1 mg/mL) was added to each well. XVIVO-15media was added to a final volume of 2.0 mL per well. Unless otherwiseindicated, lentiviruses also expressed the sgRNAs. Plates were spun at600×g for 1.5 hours at room temperature. After 18 hours (Day 2), cellswere washed and seeded at 1×10⁶ cells/mL in X-VIVO 15, 10 ng/mL IL2+T-cell activators.

Retroviral transduction of murine T cells: Where indicated, gRNAs wereintroduced into Cas9 transgenic T cells by Retroviral transduction.Murine T-cells activated 24 hours prior were seeded at 3×10⁶ cells perwell in a 6 well plate coated with 5 μg/mL RetroNectin (Takara ClontechCatalog #T100B), in 2 mL Retrovirus supernatant with 5 μg/mL protaminesulfate and 2 ng/mL of Recombinant Mouse IL-2. Retroviruses express thegRNAs and a surface selection marker (hCD2 or CD90.1). Plates were spunat 600×g for 1.5 hours at room temperature. After 18 hours (Day 2),cells were washed and seeded at 1×10⁶ cells/mL in complete T cell mediasupplemented with 2 ng/mL of Recombinant Mouse IL-2. 24 hours afterinfection, cells were washed and cultured in complete T cell mediasupplemented with IL-2. After 24 hours, activation beads were removedand infected cells were purified using EasySep Human CD2 PositiveSelection Kit (StemCell Catalog #18657) or EasySep Mouse CD90.1 PositiveSelection Kit (StemCell Catalog #18958). Edited murine CD8 T werecultured at 1×10⁶ cells/mL in complete T cell media supplemented withIL-2 for an additional 1-4 days.

Electroporation of human T cells: 3 days after T cell activation, Tcells were harvested and resuspended in nucleofection buffer (18%supplement 1, 82% P3 buffer from the Amaxa P3 primary cell4D-Nuclefector X kit S) at a concentration of 100×10⁶ cells/mL. 1.5 μLof sgRNA/Cas9 RNP complexes (containing 120 pmol of crRNA:tracrRNAduplex and 20 pmol of Cas9 nuclease) and 2.1 μL (100 pmol) ofelectroporation enhancer were added per 20 μL of cell solution. 25 μL ofthe cell/RNP/enhancer mixture was then added to each electroporationwell. Cells were electroporated using the Lonza electroporator with the“EO-115” program. After electroporation, 80 μL of warm X-VIVO 15 mediawas added to each well and cells were pooled into a culture flask at adensity of 2×10⁶ cells/mL in X-VIVO 15 media containing IL-2 (10 ng/mL).On Day 4, cells were washed, counted, and seeded at densities of50-100×10⁶ cells/L in X-VIVO 15 media containing IL-2 (10 ng/mL) inG-Rex6M well plates or G-Rex100M, depending on the number of cellsavailable. On Days 6 and 8, 10 ng/mL of fresh recombinant human IL-2 wasadded to the cultures.

Electroporation of mouse T cells: Murine T-cells activated 48 hoursprior were harvested, activation beads were removed and cells werewashed and resuspended in Neon nucleofection buffer T. Up to 2×10⁶ cellsresuspended in 9 uL Buffer T and 20×10⁶ cells resuspended in 90 uLBuffer T can be electroporated using Neon™ 10-μL tip and Neon™ 100-μLtip respectively. gRNA/Cas9 RNP complexes or ZFN mRNAs (1 μL per 10 μLtip or 10 μL per 100 μL Tip) and 10.8 μM electroporation enhancer (2 μLper 10 μL Tip or 20 μL per 100 μL Tip) were added to the cells. T-cellsmixed with gRNA/Cas9 RNP complexes or ZFN mRNAs were pipeted into theNeon™ tips and electroporated using the Neon Transfection System (1700V/20 ms/1 pulses). Immediately after electroporation, cells weretransferred into a culture flask at a density of 1.6×10⁶ cells/mL inwarm complete T cell media supplemented with 2 ng/mL of RecombinantMouse IL-2. Edited murine CD8 T cells were further cultured at 1×10⁶cells/mL in complete T cell media supplemented with IL-2 for anadditional 1-4 days.

Purification and characterization of engineered T cells: 10 days after Tcell activation, cells were removed from the culture flasks, and edited,engineered receptor-expressing CD8⁺ T cells were purified. Expression ofthe engineered receptor can be determined by antibody staining, e.g.,antibodies for Vβ12 for DMF4 TCR or Vβ13/13.1 for NY-ESO-1 or 1G4).Further determination of editing of target genes can be assessed by FACSanalysis of surface proteins (e.g., CD3), western blot of the targetprotein, and/or TIDE/NGS analysis of the genomic cut-site. Purifiedcells can then be slow-frozen in Cryostor CS-10 (Stemcell TechnologiesCat #07930) using Coolcell Freezing containers (Corning), and storedlong term in liquid nitrogen for future use.

Example 2: Characterization of Edited, Receptor-Engineered T Cells

Experiments were performed in which edited receptor-expressing cellswere purified based on cell surface expression of CD3. Prior toengineering, CD8+ T cells express CD3 molecules on the cell surface aspart of a complex that includes the TCR α/β chains (FIG. 4A). The Tcells were transduced with a lentivirus expressing a CAR, a guide RNAtargeting the TRAC gene, and a guide RNA targeting the B2M gene, whichwas used to assess the editing of non-TCR genes as a proxy for targetgene editing. Following lentiviral transduction and Cas9 mRNAelectroporation, successfully transduced and edited T cells demonstratea loss of surface CD3 expression due to editing of the TRAC gene and aloss of HLA-ABC expression due to the editing of the B2M gene (FIG. 4B).CD3-expressing cells were removed from the bulk population (FIG. 4B)using the EasySep human CD3-positive selection kit (StemCell Tech Cat#18051). Cells were then subjected to two rounds of negative magneticselection for CD3. This process yielded highly purified CD3-negative Tcells expressing (FIG. 4C). Staining with a recombinant CD19-Fc reagent(which binds CD19 CAR) demonstrated that edited cells show surfaceexpression of the CD19 CAR, whereas unedited cells do not (FIG. 4D).Similar experiments were performed with CD45 and B2M targeting sgRNAs.Cas9 editing activity in T cells was confirmed by assessing CD45 and B2Mexpression by flow cytometry was assessed 96 hours later, and efficientCas9 function is indicated by a loss of CD45 expression on the surfaceof the T cells as determined by FACS. Co-electroporation with Cas9 mRNAand Cas9 RNPs led to substantial editing at the CD45 and B2M loci, with66.3% of the cells exhibiting dual editing.

Target editing was performed as described in the above examples and theediting of a single exemplary gene, CBLB, was confirmed using both theTracking of Indels by Decomposition (TIDE) analysis method and westernblot analysis. TIDE quantifies editing efficacy and identifies thepredominant types of insertions and deletions (indels) in the DNA of atargeted cell pool.

Genomic DNA (gDNA) was isolated from edited T cells using the QiagenBlood and Cell Culture DNA Mini Kit (Cat #: 13323) following the vendorrecommended protocol and quantified. Following gDNA isolation, PCR wasperformed to amplify the region of edited DNA using locus-specific PCRprimers (F: 5′-CCACCTCCAGTTGTTGCATT-3′ (SEQ ID NO: 32); R:5′-TGCTGCTTCAAAGGGAGGTA-3′ (SEQ ID NO: 33). The resulting PCR productswere run on a 1% agarose gel, extracted, and purified using the QIAquickGel Extraction Kit (Cat #: 28706). Extracted products were sequenced bySanger sequencing and Sanger sequencing chromatogram sequence files wereanalyzed by TIDE. In CBLB-edited T cells edited by methods using eithergRNA/Cas9 RNP complexes or Cas9 mRNA introduced with gRNA expressinglentivirus (in each case, the CBLB gRNA used is SEQ ID NO: 288), theresulting TIDE analysis confirmed editing of the CBLB target gene. Inaddition to TIDE, depletion of CBLB protein levels were confirmed bywestern blot using an anti-CBLB antibody (SCT Cat #9498). The data isprovided in FIGS. 5A and 5B and FIG. 6.

Another method by which editing of a gene is assessed is by nextgeneration sequencing. For this method, genomic DNA (gDNA) was isolatedfrom edited T cells using the Qiagen Blood and Cell Culture DNA Mini Kit(Cat #: 13323) following the vendor recommended protocol and quantified.Following gDNA isolation, PCR was performed to amplify the region ofedited genomic DNA using locus-specific PCR primers containing overhangsrequired for the addition of Illumina Next Generation sequencingadapters. The resulting PCR product was run on a 1% agarose gel toensure specific and adequate amplification of the genomic locus occurredbefore PCR cleanup was conducted according to the vendor recommendedprotocol using the Monarch PCR & DNA Cleanup Kit (Cat #: T1030S).Purified PCR product was then quantified, and a second PCR was performedto anneal the Illumina sequencing adapters and sample specific indexingsequences required for multiplexing. Following this, the PCR product wasrun on a 1% agarose gel to assess size before being purified usingAMPure XP beads (produced internally). Purified PCR product was thenquantified via qPCR using the Kapa Illumina Library Quantification Kit(Cat #: KK4923) and Kapa Illumina Library Quantification DNA Standards(Cat #: KK4903). Quantified product was then loaded on the IlluminaNextSeq 500 system using the Illumina NextSeq 500/550 Mid Output ReagentCartridge v2 (Cat #: FC-404-2003). Analysis of produced sequencing datawas performed to assess insertions and deletions (indels) at theanticipated cut site in the DNA of the edited T cell pool.

Example 3: Identification of Adoptive T Cell Transfer Therapy TargetsThrough an OT1/B16-Ova CRISPR/Cas9 Functional Genomic Screen

Experiments were performed to identify targets that regulateaccumulation of T cells in tumors. A pooled CRISPR screen was performedin which a pool of sgRNAs, each of which target a single gene, wereintroduced into a population of tumor-specific T cells such that eachcell in the population comprised a single sgRNA targeting a single gene.To determine the effect of a particular gene on the accumulation of Tcells in tumor samples, the frequency of each sgRNA in the population ofT cells was determined at the beginning of the experiment and comparedto the frequency of the same sgRNA at a later time-point in theexperiment. The frequency of sgRNAs targeting genes that positivelyregulate T cell accumulation in tumor samples (e.g., genes thatpositively-regulate T cell proliferation, viability, and/or tumorinfiltration) is expected to increase over time, while the frequency ofsgRNAs targeting genes that negatively regulate T cell accumulation intumor samples (e.g., genes that negatively-regulate T cellproliferation, viability, and/or tumor infiltration) is expected todecrease over time.

Pooled CRISPR screens were performed with CD8⁺ T cells derived from Cas9expressing OT1 mice according to methods described in Example 1. Thepooled sgRNA library was introduced to purified OT1 CD8⁺ T cellscultured in vitro to generate a population of edited CD8⁺ T cells. Afterin vitro engineering, the edited OT1 CD8⁺ T cells were intravenously(iv) administered to B16/Ova tumor-bearing, C56BL/6 mice. After in vivoexpansion, organs were harvested and CD45+ cells were enriched. GenomicDNA from the isolated CD45+ cells was isolated using Qiagen DNAextraction kits. The sgRNA library was then amplified by PCR andsequenced using Illumina next-generation sequencing (NGS).

The distribution and/or frequency of each sgRNA in samples harvestedfrom tumor-bearing mice was analyzed and compared to the distributionand/or frequency of each sgRNA in the initial T cell population.Statistical analyses were performed for each individual sgRNA toidentify guides that were significantly enriched in T cell populationsharvested from tumor bearing mice and to assign an enrichment score toeach of the guides. Enrichment scores for individual sgRNA targeting thesame gene were aggregated to identify target genes that had a consistentand reproducible effect on T cell accumulation across multiple sgRNAsand across multiple OT1 donor mice. The results of these experiments areshown below in Table 12. Percentiles in Table 12 were calculated usingthe following equation: percentile score=1-(gene enrichment rank/totalnumber of genes screened).

TABLE 12 Target Gene Percentile Scores Target Name Percentile ScoreIKZF1 0.995 NFKBIA 0.986 GATA3 0.993 BCL3 0.698 IKZF3 0.995 SMAD2 0.978TGFBR1 0.991 TGFBR2 0.987 TNIP1 0.991 TNFAIP3 0.998 IKZF2 0.622 TANK0.83 PTPN6 0.782 BCOR 0.720 CBLB 0.999 NRP1 0.826 HAVCR2 0.86 LAG3 0.82BCL2L11 0.9928 CHIC2 0.997 FLI1 0.999 PCBP1 0.997 PBRM1 0.944 WDR6 0.953E2F8 0.867 SERPINA3 0.822 SEMA7A 0.78 DHODH 0.990 UMPS 0.989 ZC3H12A0.999 MAP4K1 0.842 NR4A3 0.997

Example 4: Identification of Adoptive T Cell Transfer Therapy TargetsThrough In Vitro CAR-T and CRISPR/Cas9 Functional Genomic Screens

Experiments were performed to identify targets that regulateaccumulation of CAR-T cells tumor samples. A pooled, genome-wide CRISPRscreen was performed in which a pool of sgRNAs, each of which target asingle gene, was introduced into a population of tumor-specific humanCAR-T cells, such that each cell in the population comprised a singlesgRNA targeting a single gene. To determine the effect of a particulargene in CAR-T cell accumulation in tumor samples, the frequency of eachsgRNA in the population of CAR-T cells was determined at the beginningof the experiment and compared to the frequency of the same sgRNA at alater time-point in the experiment. The frequency of sgRNAs targetinggenes that positively regulate CAR-T cell accumulation in tumor samples(e.g., genes that positively-regulate T cell proliferation, viability,and/or tumor infiltration) is expected to increase over time, while thefrequency of sgRNAs targeting genes that negatively regulate CAR-T cellaccumulation in tumor samples (e.g., genes that negatively-regulate Tcell proliferation, viability, and/or tumor infiltration) is expected todecrease over time.

In vitro screens were performed using CAR-T cells specific for humanCD19. Pooled sgRNA libraries were introduced to the CD19 CARTs asdescribed above and cells were electroporated with Cas9 mRNA asdescribed in Example 1 to generate a population of Cas9-edited CD19CARTs. The edited CD19 CARTs were then co-cultured with an adherentcolorectal carcinoma (CRC) cell line engineered to express CD19 or aBurkitt's lymphoma cell line expressing endogenous CD19. CARTs wereharvested at various time points throughout the co-culture period andcell pellets were frozen down. Genomic DNA (gDNA) was isolated fromthese cell pellets using Qiagen DNA extraction kits and sequenced usingIllumina next-generation sequencing.

The distribution and/or frequency of each sgRNA in the aliquots takenfrom the CART/tumor cell co-culture was analyzed and compared to thedistribution and/or frequency of each sgRNA in the initial edited CAR-Tcell population. Statistical analyses were performed for each individualsgRNA to identify sgRNAs that were significantly enriched in CAR-T cellpopulations after tumor cell co-culture and to assign an enrichmentscore to each of the guides. Enrichment scores for individual sgRNA thattarget the same gene were aggregated to identify target genes that havea consistent and reproducible effect on CAR-T cell accumulation in tumorsamples across multiple sgRNAs and CAR-T cell population. Targets wereranked and called for further investigation based on percentile. Theresults of these experiments are shown below in Table 13. Percentiles inTable 13 were calculated using the following equation: percentilescore=1−(gene enrichment rank/total number of genes screened).

TABLE 13 Target Gene Percentile Scores Target Name Percentile ScoreIKZF1 0.999 IKZF3 0.962 TGFBR1 0.778 TNIP1 0.64 TNFAIP3 0.791 FOXP30.866 IKZF2 0.907 TANK 0.93 PTPN6 0.707 BCOR 0.999 CBLB 0.989 BCL2L110.93 CHIC2 0.71 WDR6 0.962 E2F8 0.971 DHODH 0.763

Example 5: Identification of Adoptive T Cell Transfer Therapy TargetsThrough an In Vivo CAR-T/Tumor CRISPR/Cas9 Functional Genomic Screen

Experiments were performed to identify targets that regulate CAR-T cellaccumulation in the presence of tumors. A pooled CRISPR screen wasperformed in which a pool of sgRNAs, each of which target a single gene,was introduced into a population of tumor-specific human CAR-T cellssuch that each cell in the population comprised a single sgRNA targetinga single gene. To determine the effect of a particular gene in CAR-Tcell accumulation in tumor samples, the frequency of each sgRNA in thepopulation of CAR-T cells was determined at the beginning of theexperiment and compared to the frequency of the same sgRNA at a latertime-point in the experiment. The frequency of sgRNAs targeting genesthat positively regulate CAR-T cell accumulation in tumor samples (e.g.,genes that positively-regulate T cell proliferation, viability, and/ortumor infiltration) is expected to increase over time, while thefrequency of sgRNAs targeting genes that negatively regulate CAR-T cellaccumulation in tumor samples (e.g., genes that negatively regulate Tcell proliferation, viability, and/or tumor infiltration) is expected todecrease over time.

In vivo screens performed in two separate subcutaneous xenograft models:a Burkitt lymphoma model and a colorectal cancer (CRC) model. For theBurkitt model, 1×10⁶ Burkitt lymphoma tumor cells in Matrigel weresubcutaneously injected into the right flank of 6-8 week old NOD/SCIDgamma (NSG) mice. Mice were monitored, randomized, and enrolled into thestudy 13 days post-inoculation, when tumors reached approximately 200mm³ in volume. For the CRC model, CRC cells were engineered to expressCD19, and 5×10⁶ tumor cells in Matrigel were subcutaneously injectedinto the right flank of 6-8 week old NSG mice. Mice were monitored,randomized, and enrolled into the study 12 days post-inoculation whentumors reached approximately 200 mm³ in volume. Cas9-engineered CD19CAR-T cells were administered iv via the tail vein at 3×10⁶ and10×10⁶/mouse (3M and 10M). Tumors were collected 8 to 10 days post-CAR-Tinjection and frozen in liquid nitrogen. These tissues were laterdissociated and processed for genomic DNA extraction.

The distribution and/or frequency of each sgRNA in the genomic DNAsamples taken at study end was analyzed and compared to the distributionand/or frequency of each sgRNA in the initial edited-CAR-T cellpopulation. Statistical analyses were performed for each individualsgRNA to identify sgRNAs were are significantly enriched in genomic DNAsamples taken at study end and to assign an enrichment score to each ofthe guides. Enrichment scores for individual sgRNA that target the samegene were aggregated to identify target genes that have a consistent andreproducible effect on CAR-T cell abundance across multiple sgRNAs andCAR-T cell populations. Targets were ranked and called for furtherinvestigation based on percentile. The results of these experiments areshown below in Table 14. Percentiles in Table 14 were calculated usingthe following equation: percentile score=1−(gene enrichment rank/totalnumber of genes screened).

TABLE 14 Target Gene Percentile Scores Target Name Percentile ScoreNFKBIA 0.95 SMAD2 0.816 FOXP3 0.92 IKZF2 0.895 TANK 0.923 PTPN6 0.979CBLB 0.958 PPP2R2D 0.926 NRP1 0.795 HAVCR2 0.992 LAG3 0.97 TIGIT 0.916CTLA4 0.884 BCL2L11 0.776 RBM39 0.94 E2F8 0.968 CALM2 0.902 SERPINA30.907 SEMA7A 0.918

Example 6: Validation of Single-Edited Adoptively Transferred T Cells ina Murine OT1/B16-Ova Syngeneic Tumor Model

Targets with percentile scores of 0.6 or greater in Examples 3-5 wereselected for further evaluation in a single-guide format to determinewhether editing a target gene in tumor-specific T cells conferred anincrease in anti-tumor efficacy in a murine OT1/B16-Ova subcutaneoustumor model. Evaluation of exemplary targets is described herein,however these methods can be used to evaluate any of the potentialtargets described above.

Anti-tumor efficacy of single-edited T cells was evaluated in mice usingthe B16-Ova subcutaneous syngeneic tumor model, which is sensitive totreatment with anti-PD1 antibodies. Briefly, 6-8 week old femaleC57BL/6J mice from Jackson labs were injected subcutaneously with0.5×10⁶ B16-Ova tumor cells. When tumors reached a volume ofapproximately 100 mm³ mice were randomized into groups of 10 andinjected intravenously with edited mouse OT1 CD8+ T cells via tail vein.Prior to injection, the OT1 T cells were edited by electroporation withgRNA/Cas9 RNP complexes comprising (i) a control gRNA; (ii) a singleMap4k1-targeting gRNA, (iii) a single Zc3h12a-targeting gRNA; (iv) asingle Cblb-targeting gRNA; (v) a single Nr4a3-targeting gRNA or; (vi) asingle PD1-targeting gRNA. To generate a population of tumor-specificCD8⁺ T cells with edited target genes, spleens from female OT1 mice wereharvested and CD8 T cells were isolated and edited as described inExample 1. The edited OT1 CD8⁺ T cells were then administeredintravenously to B16-Ova tumor-bearing C56BL/6 mice. Body weight andtumor volume were measured at least twice per week. Tumor volume wascalculated as mean and standard error of the mean for each treatmentgroup. The percentage tumor growth inhibition (TGI) was calculated usingmean tumor volumes (TV) according to the following formulas:%TGI=(TV-Target_(final)−TV-Target_(Initial))/(TV-Control_(final)−TV-Control_(Initial)),

where TV=mean tumor volume, final for Cblb TGI=Day 18 post-T celltransfer, final for Zc3h12a, Nr4a3, and Map4k1=Day 14 post-T celltransfer, and initial=Day 0 (i.e., day of T cell transfer).

Results of Cblb-edited T cells are shown in FIG. 7A. These datademonstrate that editing of the Cblb gene in T cells leads to anti-tumorefficacy with a TGI of 85% at day 18. Results of Zc3h12a-edited T cellsare shown in FIG. 7B. These data demonstrate that editing of the Zc3h12agene in T cells enhances anti-tumor efficacy of the T cells with a TGIof 106% at day 14. Data from an additional experiment withZc3h12a-edited cells are shown in FIG. 7C. Results of Map4k1-edited Tcells are shown in FIG. 7D. These data demonstrate that editing of theMap4k1 gene in T cells enhances anti-tumor efficacy of the T cells witha TGI of 72% at day 14. Results of Nr4a3-edited T cells are shown inFIG. 7E. These data demonstrate that editing of the Nr4a3 gene in Tcells enhances anti-tumor efficacy of the T cells with a TGI of 56% atday 14.

Example 7: Validation of Single-Edited Adoptively Transferred T Cells ina Murine OT1/B16-Ova Syngeneic Tumor Model in Combination with Anti-PD1Therapy

Further experiments were performed to assess the effect of Map4k1-editedcells in combination with anti-PD1 therapy. 6-8 week old female C57BL/6Jmice from Jackson labs were injected subcutaneously with 0.5×10⁶ B16-Ovatumor cells. When tumors reached a volume of approximately 100 mm³ micewere randomized into groups of 10 and injected intravenously with editedmouse OT1 CD8+ T cells via tail vein. Prior to injection these cellswere edited with either a control guide or a single guide editing forthe Map4k1 gene. The editing efficiency of the gRNA/Cas9 complextargeting Map4k1 gene was determined to be 82% using the NGS method. Atthe time of T cell transfer, designated cohorts were also initiatedtreatment with 2.5 mg/kg of anti-mouse PD1 antibody (clone RMP1-14,BioXcell), injected 3× week for the duration of the study. Body weightand tumor volume was measured at least twice per week. Tumor volume wascalculated as mean and standard error of the mean for each treatmentgroup. The percentage tumor growth inhibition (TGI) was calculated usingthe mean tumor volume from the Map4k1 group on day 14−day 1/mean tumorvolume from control treated group on day 14-day 1 where day 1 is the dayof edited mouse OT1 CD8+ T cell transfer. Results of this experiment areshown in FIG. 8A.

A similar experiment was conducted using Nr4a3-edited cells. The editingefficiency of the gRNA/Cas9 complex targeting the Nr4a3 gene wasdetermined to be 28% using the NGS method. The percentage tumor growthinhibition (TGI) was calculated using the mean tumor volume from theNr4a3 group on day 14−day 1/mean tumor volume from control treated groupon day 14-day 1 where day 1 is the day of edited mouse OT1 CD8+ T celltransfer. Results of this experiment are shown in FIG. 8B.

Similar experiments are performed to assess the effect of Zc3h12a-editedcells in combination with anti-PD1 therapy on anti-tumor efficacy.

Example 8: Validation of Single-Edited Adoptively Transferred T Cells ina Murine PMEL and MC38/gp100 Syngeneic Tumor Model

Targets with percentile scores of 0.6 or greater in Examples 3-5 wereselected for further evaluation in a single-guide format to determinewhether editing a target gene in tumor-specific T cells conferred anincrease in anti-tumor efficacy in a murine MC38gp100 subcutaneoussyngeneic tumor model of colorectal cancer (which is insensitive totreatment with anti-PD1 antibodies). Evaluation of exemplary targets isdescribed herein, however these methods can be used to evaluate any ofthe potential targets described above.

Briefly, 6-8 week old female C57BL/6J mice from Jackson labs wereinjected subcutaneously with 1×10⁶ MC38gp100 tumor cells. Prior toinjection, the T cells were edited by electroporation with gRNA/Cas9 RNPcomplexes comprising (i) a control gRNA or (ii) a singleZc3h12a-targeting gRNA. When tumors reached a volume of approximately100 mm³ mice were randomized into groups of 10 and injectedintravenously with Zc3h12a-edited mouse PMEL CD8⁺ T cells via tail vein.Body weight and tumor volume was measured at least twice per week. Tumorvolume was calculated as mean and standard error of the mean for eachtreatment group.

Results of Zc3h12a-edited T cells are shown in FIG. 9. These datademonstrate that editing of the Zc3h12a gene in T cells enhancesanti-tumor efficacy of the T cells with a TGI of 77% at day 20. Similarexperiments are performed to assess the effect of Nr4a3-edited cells andMap4k1-edited cells on anti-tumor efficacy in a PMEL/MC38 syngeneictumor model.

Example 9: Validation of Single-Edited Adoptively Transferred T Cells ina Murine B16-F10 Syngeneic Tumor Model

Additional experiments are performed to assess the effect of editingZc3h12a in the aggressive metastatic B16-F10 syngeneic tumor model withdisease manifesting as lung metastasis.

Briefly, 6-8 week old female C57BL/6J mice from Jackson labs areinjected intravenously with 0.5×10⁶ B16-F10 tumor cells. Mice areweighed and assigned to treatment groups using a randomization procedureprior to inoculation. At D3 post tumor inoculation, mice are injectedintravenously with murine PMEL CD8+ T cells via tail vein. Prior toinjection, these cells were edited by electroporation with gRNA/Cas9 RNPcomplexes comprising (i) a control gRNA; (ii) a single gRNA targetingthe PD-1 gene; or (iii) a single gRNA targeting the Zc3h12a gene. Bodyweight is monitored at least twice per week. At Day 15 post tumorinoculation (Day 12 post edited PMEL transfer), mice lungs are perfusedand fixed with 10% para-formaldehyde. After overnight fixation, lungsare transferred to 70% EtOH for further preservation. Anti-tumorefficacy can be evaluated by visually assessing the B16-F10 tumor burdenwhich can be seen as black colonies of cancer cells on the lungs.

Survival is graphed as percent (FIG. 10). As shown in FIG. 10A,Zc3h12a-edited PMEL T cells were able to convey increased survival incomparison to control and PD1-edited PMEL T cells in a B16F10 lung metmodel. Similar experiments are performed to assess the effects ofNr4a3-edited and Map4k1-edited cells in a B16-F10 syngeneic tumor model.

Example 10: Validation of Single-Edited Adoptively Transferred T Cellsin a Murine OT1/EG7-Ova Subcutaneous Syngeneic Tumor Model

Anti-tumor efficacy of Zc3h12a was further evaluated in mice using theEg7-Ova subcutaneous syngeneic tumor model. 6-8 week old female C57BL/6Jmice from Jackson labs were injected subcutaneously with 1×10⁶ Eg7-Ovatumor cells. When tumors reached a volume of approximately 100 mm³ micewere randomized into groups of 10 and injected intravenously with editedmouse OT1 CD8+ T cells via tail vein. Prior to injection these cellswere edited with either a control guide or a single guide editing forthe Zc3h12a gene. Body weight and tumor volume was measured at leasttwice per week. Tumor volume was calculated as mean and standard errorof the mean for each treatment group.

The percentage tumor growth inhibition (TGI) was calculated using meantumor volumes (TV) according to the following formulas:%TGI=(TV-Target_(final)−TV-Target_(Initial))/(TV-Control_(final)−TV-Control_(Initial)),

where TV=mean tumor volume, final TGI=Day 14 post-T cell transfer, andinitial=Day 0 (i.e., day of T cell transfer).

These data demonstrate that editing of the Zc3h12a in T cells enhancesanti-tumor efficacy of the T cells with a TGI of 88% as shown in in FIG.11. Similar experiments are performed to assess the effect ofNr4a3-edited cells and Map4k1-edited cells on anti-tumor efficacy in aOT1/EG7OVA subcutaneous syngeneic tumor model.

Example 11: Validation of Single-Edited Adoptively Transferred T Cellsin the A375 Xenograft Tumor Model

Targets with percentile scores of 0.6 or greater in Examples 3-5 wereselected for further evaluation in a single-guide format to determinewhether editing a target gene in tumor-specific T cells conferred anincrease in anti-tumor efficacy in the A375 xenograft tumor model.Evaluation of exemplary targets is described herein, however thesemethods can be used to evaluate any of the potential targets describedabove.

Briefly, 6-12 week old NSG mice from Jackson labs were injectedsubcutaneously with 5×10⁶ A375 cells. When tumors reached a volume ofapproximately 200 mm³, mice were randomized into groups of 8 andinjected intravenously with 18.87×10⁶ for CBLB-edited NY-ESO-1-specificTCR transgenic T cells or 5.77×10⁶ ZC3H12A-edited NY-ESO-1-specific TCRtransgenic T cells via tail vein. Body weight and tumor volume wasmeasured at least twice per week. Tumor volume was calculated as meanand standard error of the mean for each treatment group and efficacy wasevaluated as a decrease in mean tumor volume and an increase in survivalcompared to control edited cells. The percentage tumor growth inhibition(TGI) was calculated using the mean tumor volume from the ZC3H12A groupon day 19−day 0/mean tumor volume from control treated group on day19−day 0 where day 0 is the day of human TCR transgenic T cell transfer.

Results of these experiments are shown in FIG. 12. These datademonstrate that editing of the CBLB (FIG. 12A) and ZC3H12A (FIG. 12B)gene in T cells leads to anti-tumor efficacy in an A375 xenograft model.Similar experiments are performed to assess the efficacy ofMAP4K1-edited T cells and NR4A3-edited T cells in the A375 xenograftmodel.

Example 12: Validation of Single-Edited Adoptively Transferred CD19CAR-T Cells in a Raji Xenograft Model

The anti-tumor efficacy of editing MAP4K1 and NR4A3 in 1^(st) generationCD19 CAR-T cells was evaluated in mice using the Raji subcutaneous cellderived xenograft tumor model. Raji cells are a human lymphoma cell linethat are known to be insensitive to treatment with anti-PD1 antibodies.

Briefly, 6-8 week old female NSG mice from Jackson labs were injectedsubcutaneously with 3×10⁶ Raji tumor cells. When tumors reached a volumeof approximately 200 mm³ mice were randomized into groups of 5 andinjected intravenously with edited human CD19 CART cells via tail vein.Prior to injection the adoptively transferred cells were edited witheither a control guide or a guide editing for MAP4K1 or NR4A3. Using theNGS method, the editing efficiency of the gRNA/Cas9 complex targetingthe MAP4K1 gene and NR4A3 were determined to be 83% and 71%,respectively. Body weight and tumor volume was measured at least twiceper week. Tumor volume was calculated as mean and standard error of themean for each treatment group. The results demonstrate that editing ofMAP4K1 and NR4A3 in an adoptive T cell transfer model leads to increasedefficacy, with a 65% TGI for MAP4K1 (FIG. 13A) and 81% TGI for NR4A3(FIG. 13B). Similar experiments will be performed to assess theanti-tumor efficacy of ZC3H12A-edited 1^(st) generation CD19 CAR-T cells(human) in the Raji cell-derived xenograft subcutaneous tumor model.

Example 13: Screen for Dual-Edit Combinations

A double sgRNA library was constructed in a retroviral backbone. Thelibrary consisted of two U6 promoters (one human and one mouse), eachdriving expression of a single guide RNA (guide+tracr, sgRNA). Theguides were cloned as pools to provide random pairings between guides,such that every sgRNA would be paired with every other sgRNA. The finaldouble guide library was transfected into Phoenix-Eco 293T cells togenerate murine ecotropic retrovirus. TCR transgenic OT1 cellsexpressing Cas9 were infected with the sgRNA-expressing virus to editthe two loci targeted by each of the sgRNAs. The edited transgenicT-cells were then transferred into mice bearing >400 mm³ B16-Ova tumorsallografts. After two weeks, the tumors were excised and thetumor-infiltrating T-cells were purified by digesting the tumors andenriching for CD45+ cells present in the tumors. Genomic DNA wasextracted from CD45+ cells using a Qiagen QUIAamp DNA blood kit and theretroviral inserts were recovered by PCR using primers corresponding tothe retroviral backbone sequences. The resulting PCR product were thensequenced to identify the sgRNAs present in the tumors two weeks aftertransfer. The representation of guide pairs in the final isolated cellpopulations was compared to the initial plasmid population and thepopulation of infected transgenic T-cells before injection into themouse. The frequency of sgRNA pairs that improved T-cells fitness and/ortumor infiltration were expected to increase over time, whilecombinations that impaired fitness were expected to decrease over time.Table 15 below shows the median fold change of sgRNA frequency in thefinal cell population compared to the sgRNA frequency in the initialcell population transferred in vivo.

TABLE 15 sgRNA frequency in Combination Screen GeneA GeneBAvg(Tmedian.Ifoldch.all) CBLB CBLB 0.17 CBLB CTLA4 0.21 CBLB LAG3 0.08CBLB Olfr1389 0.03 CBLB Olfr453 0.04 CBLB TGFBR1 0.15 CBLB TGFBR2 0.75CBLB TIGIT 0.31 CBLB ZAP70 0 Havcr2 Havcr2 0.02 Havcr2 LAG3 0.01 Havcr2Olfr1389 0 Havcr2 Olfr453 0.01 Havcr2 PDCD1 0.02 LAG3 Olfr1389 0 LAG3Olfr453 0.02 LAG3 PDCD1 0.02 Olfr1389 Olfr1389 0.01 Olfr1389 Olfr453 0Olfr1389 PDCD1 0.02 Olfr453 Olfr453 0.01 Olfr453 PDCD1 0.01 PDCD1 CTLA40.59 PDCD1 LAG3 0.02 PDCD1 PDCD1 0.02 PDCD1 TGFBR1 0.02 PDCD1 TGFBR20.07 PDCD1 TIGIT 0.02 PDCD1 ZAP70 0 TGFBR1 CTLA4 0.01 TGFBR1 LAG3 0TGFBR1 TGFBR1 0.06 TGFBR1 TGFBR2 0.07 TGFBR1 TIGIT 0.03 TGFBR1 ZAP70 0CBLB ZC3H12A 8.9 Havcr2 ZC3H12A 2.1 Olfr1389 ZC3H12A 0.78 Olfr453ZC3H12A 1.58 PDCD1 ZC3H12A 1.33 TGFBR1 ZC3H12A 3.33 Tigit ZC3H12A 2.53ZAP70 ZC3H12A 0.01 ZC3H12A CTLA4 0.61 ZC3H12A LAG3 0.73 ZC3H12A ZAP700.01 ZC3H12A ZC3H12A 1.14

Example 14: Validation of Dual-Edited CD19 CAR-T Cells in Raji XenograftModel

Targets were further evaluated in combination studies to determinecombinations of edited genes that increased anti-tumor efficacy of Tcells in xenograft tumor models. Evaluation of exemplary targets isdescribed herein, however these methods can be used to evaluate any ofthe potential targets described above

As an example of a combination effect for anti-tumor efficacy ofediting, CBLB and BCOR were edited, either independently or together, in1^(st) generation CD19 CAR-T cells and evaluated in mice using the Rajisubcutaneous cell derived xenograft tumor model. Raji cells are alymphoma cell line that are known to be insensitive to treatment withanti-PD1 antibodies. 6-8 week old female NSG mice from Jackson labs wereinjected subcutaneously with 3×10⁶ Raji tumor cells. When tumors reacheda volume of approximately 200 mm³ mice were randomized into groups of 5and injected intravenously with edited human CD19 CART cells via tailvein. Prior to injection, the adoptively transferred cells were editedby electroporation with gRNA/Cas9 RNP complexes comprising (i) a controlgRNA; (ii) single gRNA targeting CBLB; (iii) a single gRNA targetingBCOR; or (iv) 2 gRNAs targeting CBLB and BCOR. Body weight and tumorvolume were measured at least twice per week. Tumor volume wascalculated as mean and standard error of the mean for each treatmentgroup. As shown in FIG. 14, when compared to a control guide, adoptivetransfer of BCOR/CBLB dual-edited human CD19 CART cells, with targetgenes edited either alone or together as indicated, resulted in ananti-tumor response in the subcutaneous Burkitt's Lymphoma Raji mousemodel. The anti-tumor efficacy was greater when both targets were editedin combination as compared to either target alone or as compared to acontrol guide. Similar experiments are performed to assess the efficacyof ZC3H12A/CBLB dual-edited T cells, MAP4K1/CBLB dual-edited T cells,and NR4A3I CBLB dual-edited T cells in the CD19 CAR-T Raji cellxenograft model.

Example 15: Double-Editing of BCOR and CBLB in CAR-Ts Leads to EnhancedAccumulation and Cytokine Production in the Presence of Tumor

1^(st) generation CD19 CAR-Ts were generated from human CD8 T cells, anda negative control gene, BCOR, CBLB, or both BCOR and CBLB were editedby electroporation using guide RNAs complexed to Cas9 in an RNP format.CD19 CAR-Ts were co-cultured with Raji Burkitt's Lymphoma cells in vitroat a 1:0, 0.3:1, 1:1, 3:1 and 10:1 ratio. After 24 hours, total cellcounts of CAR-T cells were determined, and supernatants saved forcytokine analyses. As shown in FIG. 15, BCOR and BCOR+CBLB-edited CARTsdemonstrated 30% greater accumulation compared to either control orCBLB-edited CARTs, demonstrating that editing of the BCOR confers anenhanced ability of the CAR-T cells to accumulate in the presence of atumor. Further, CBLB and CBLB+BCOR-edited CARTs produced 10-fold or moreIL-2 (FIG. 16) and IFNγ (FIG. 17) compared to either control-editedCARTs, demonstrating that editing of CBLB resulted in enhanced CAR-Tcell production of cytokines known to increase overall T cell fitnessand functional ability. The increased production of IL-2 by CD8 T cellsis surprising as these cells typically do not produce IL-2. These datademonstrate that, in some instances, production of CAR-T cells withenhanced effector functions requires editing of multiple genes. Forexample, in this example, the production of CAR-T cells thatdemonstrated both enhanced accumulation in the presence of a tumor andenhanced production of IL-2 and IFNγ cytokines required editing of bothBCOR and CBLB genes.

Example 16: Validation of Dual-Edited, Adoptively Transferred T Cells ina Murine OT1/B16-Ova Syngeneic Tumor Model

Targets were further evaluated in combination studies to determinecombinations of edited genes that increased anti-tumor efficacy of Tcells in a murine OT1/B16-Ova subcutaneous syngeneic tumor model.Evaluation of exemplary targets is described herein, however thesemethods can be used to evaluate any of the potential targets describedabove.

Anti-tumor efficacy of Zc3h12a/Cblb dual-edited T cells was evaluated inmice using the B16Ova subcutaneous syngeneic tumor model. Briefly, 6-8week old female C57BL/6J mice from Jackson labs were injectedsubcutaneously with 0.5×10⁶ B16Ova tumor cells. When tumors in theentire cohort of mice reached an average volume of approximately 378.3mm³, the mice were randomized into groups and injected intravenouslywith edited murine OT1 CD8+ T cells via tail vein. Prior to injection,these cells were edited by electroporation with gRNA/Cas9 RNP complexescomprising (i) a control gRNA; (ii) a single gRNA targeting the Cblbgene; (iii) a single gRNA targeting the Zc3h12a gene; or (iv) 2 gRNAstargeting the Cblb and Zc3h12a genes. Editing efficiency of thegRNA/Cas9 complex targeting the Cblb and Zc3h12a genes was determined tobe 82% and 80% respectively, assessed using the NGS method. Body weightand tumor volume were measured at least twice per week. Tumor volume wascalculated as mean and standard error of the mean for each treatmentgroup. The percentage tumor growth inhibition (TGI) was calculated usingthe mean tumor volume according to the following formula:%TGI=(TV-Target_(final)−TV-Target_(Initial))/(TV-Control_(final)−TV-Control_(Initial))*100,

-   -   where TV=mean tumor volume, final=Day 7 post-T cell transfer,        and initial=Day 0 (i.e., day of T cell transfer).

As shown in FIG. 18, transfer of Zc3h12a/Cblb dual-edited T cellsresulted in an enhanced TGI compared to transfer of Cblb single-edited Tcells or Zc3h12a single-edited T cells or Cblb single-edited T cells(Zc3h12a/Cblb TGI=107% compared to 46% and 99% for Cblb and Zc3h12asingle edits, respectively).

Similar experiments are performed to assess the efficacy of Map4k1/Cblbdual-edited T cells and Nr4a3/Cblb dual-edited T cells in the OT1/B16Ova syngeneic tumor model.

Example 17: Validation of Dual-Edited, Adoptively Transferred T Cells ina Murine PMEL/MC38-gp100 Tumor Model

Anti-tumor efficacy of Zc3h12a/Cblb dual-edited T cells, Map4k1/Cblbdual-edited T cells, and Nr4a3/Cblb dual-edited T cells is evaluated inmice using the MC38gp100 subcutaneous syngeneic tumor model. Briefly,6-8 week old female C57BL/6J mice from Jackson labs are injectedsubcutaneously with 1×10⁶ MC38gp100 tumor cells. When tumors reach avolume of approximately 100 mm³, mice are randomized into groups of 10and injected intravenously with edited murine PMEL CD8+ T cells via tailvein. Prior to injection, T cells are edited by electroporation withgRNA/Cas9 RNP complexes comprising (i) a control gRNA; (ii) a singlegRNA targeting the PD1 gene; (iii) a single gRNA targeting the Zc3h12agene; (iv) a single gRNA targeting the Cblb gene; (v) a single gRNAtargeting the Map4k1 gene; (vi) a single gRNA targeting the Nr4a3 gene;(vii) 2 gRNAs targeting both the Zc3h12a and Cblb genes; (viii) 2 gRNAstargeting both the Nr4a3 and Cblb genes; or (ix) 2 gRNAs targeting boththe Map4k1 and Cblb genes. Body weight and tumor volume are measured atleast twice per week. Tumor volume is calculated as mean and standarderror of the mean for each treatment group. The percentage TGI iscalculated using the mean tumor volume according to the followingformula:(TV-Target_(final)−TV-Target_(Initial))/(TV-Control_(final)−TV-Control_(Initial)),

where TV=mean tumor volume, final=Day 21 post-T cell transfer, andinitial=Day 0 (i.e., day of T cell transfer)

These experiments are expected to show enhanced TGI after transfer ofZc3h12a/Cblb, Map4k1/Cblb, or Nr4a3/Cblb dual-edited T cells compared totransfer of PD1 single-edited T cells, Zc3h12a single-edited T cells,Map4k1 single-edited T cells, or Nr4a3 single-edited T cells.

Example 18: Validation of Dual-Edited, Adoptively Transferred T Cells ina Murine B16-F10 Syngeneic Tumor Model

Anti-tumor efficacy of Zc3h12a/Cblb dual-edited T cells is evaluated inmice using the aggressive metastatic B16-F10 syngeneic tumor model withdisease manifesting as lung metastasis. Briefly, 6-8 week old femaleC57BL/6J mice from Jackson labs are injected intravenously with 0.5×10⁶B16-F10 tumor cells. Mice are weighed and assigned to treatment groupsusing a randomization procedure prior to inoculation. At Day 3 posttumor inoculation, mice are injected intravenously with edited murinePMEL CD8+ T cells via tail vein. Prior to injection these cells areedited by electroporation with gRNA/Cas9 RNP complexes comprising (i) acontrol gRNA; (ii) a single gRNA targeting the Zc3h12a gene; (iii) asingle gRNA targeting the Cblb gene; (iv) a single gRNA targeting theMap4k1 gene; (v) a single gRNA targeting the Nr4a3 gene; (vi) 2 gRNAstargeting each of the Map4k1 and Cblb genes; (vii) 2 gRNAs targetingeach of the Nr4a3 and Cblb genes or; (viii) 2 gRNAs targeting each ofthe Zc3h12a and Cblb genes. Editing efficiency of the gRNA/Cas9 complextargeting the aforementioned genes is assessed using the NGS method.Body weight will be monitored at least twice per week. At Day 15 posttumor inoculation (Day 12 post edited PMEL transfer), mice lungs areperfused and fixed with 10% para-formaldehyde. After overnight fixation,lungs are transferred to 70% EtOH for further preservation. Tumorefficacy is evaluated by visually assessing the B16-F10 tumor burdenwhich will be seen as black colonies of cancer cells on the lungs.

These experiments are expected to show increased anti-tumor efficacy ofdual-edited cells compared to controls and single-edited cells.

Example 19: Efficacy of Pd1/Lags Dual-Edited Transgenic T Cells in aB16-Ova Murine Tumor Model

Anti-tumor efficacy of PD-1/Lag3 dual-edited T cells was evaluated inmice using the B16Ova subcutaneous syngeneic tumor model. 6-8 week oldfemale C57BL/6J mice from Jackson labs were injected subcutaneously with0.5×10⁶ B16Ova tumor cells. When tumors in the entire cohort of micereached an average volume of approximately 485 mm³, the mice wererandomized into groups of 10 and injected intravenously with editedmouse OT1 CD8+ T cells via tail vein. Prior to injection these cellswere edited by electroporation with gRNA/Cas9 RNP complexes comprising(1) a non-targeting control gRNA; (2) a single gRNA targeting the PD1gene; (3) a single gRNA targeting the Lag3 gene; (4) 2 gRNAs, onetargeting each of the PD1 and Lag3 genes. Body weight and tumor volumewere measured at least twice per week. Tumor volume was calculated asmean and standard error of the mean for each treatment group. Thepercentage tumor growth inhibition (TGI) was calculated using thefollowing formula:% TGI=(PD1/Lag3 TV_(final))−PD1/Lag3 TV_(initial))/(ControlTV_(final)−Control TV_(initial)),

where TV=mean tumor volume, final=Day 10 and initial=day of edited mouseOT1 CD8+ T cell transfer.

The data in FIG. 19 show adoptive transfer of PD1 single-edited T cellsresulted in a TGI of 70% and adoptive transfer of Lag3 single-edited Tcells resulted in a TGI of 36%. Surprisingly, combination edits of PD1and Lag3 did not result in enhanced tumor growth inhibition anddemonstrated a TGI of 38%.

Example 20: Validation of Targets for Adoptive T Cell Transfer of TumorInfiltrating Lymphocytes

Anti-tumor efficacy of Zc3h12, Map4k1, and Nr4a3/Cblb single anddual-edited tumor infiltrating lymphocytes (TILs) are evaluated in miceusing the B16Ova subcutaneous syngeneic tumor model. Two mice cohortsare used in this experiment: a donor cohort of CD45.1 Pep Boy mice(B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ) and a recipient cohort of CD45.2C57BL/6J mice (Jackson labs), each comprised of 6-8 week old femalemice.

To generate TILs, donor CD45.1 Pep Boy mice (B6.SJL-Ptprc^(a)Pepc^(b)/BoyJ) are injected subcutaneously with 0.5×10⁶ B16-Ova cells.On Day 14 post-tumor cell inoculation, tumors are harvested to generateedited CD45.1 Tumor Infiltrating Lymphocytes (TILs) to infuse into thesecond cohort of mice. B16-OVA tumors (200-600 mm³) are harvested,diced, and reduced to a single cell suspension using the GentleMACSsystem and mouse Tumor Dissociation Kit (Miltenyi Biotech Catalog#130-096-730), according to the manufacturer's recommendations. Tumorsuspension are filtered over 70 μm cell strainers and TILs are enrichedusing CD4/CD8 (TIL) Microbeads (Miltenyi Biotech Catalog #130-116-480).Isolated TILs are cultured in 6 well plates at 1.5×10⁶ cells/mL incomplete mTIL media (RPMI+10% heat inactivated FBS, 20 mM HEPES, 100U/mL Penicillin, 100 μg/mL Streptomycin, 50 μM Beta-Mercaptoethanol, 1×Glutamx) supplemented with 3000 U/mL of recombinant human IL-2(Peprotech Catalog #200-02). On Day 3 cells are harvested, washed andresuspended in nucleofection buffer T and electroporated with gRNA/Cas9RNPs using the Neon Transfection System as described in Example 1. Afterelectroporation, TILs are cultured in 6 well plates at 1.5×10⁶ cells/mLin complete mTIL media supplemented with 3000 U/mL of recombinant humanIL-2. On Day 5 and 7, cells are resuspended in fresh complete mTIL mediasupplemented with 3000 U/mL of recombinant human IL-2 and plated inflasks at a density of 1×10⁶ cells/mL. On Day 8, cells are harvestedcounted and resuspended in PBS for injection in vivo and pellets wereprepared for gene expression analysis by qRT-PCR.

These TIL cells are edited by electroporation of gRNA/Cas9 complexescomprising (1) a non-targeting control gRNA; (2) a single gRNA targetingthe Zc3h12a gene; (3) a single gRNA targeting the Map4k1 gene; (4) asingle gRNA targeting the Nr4a3 gene; (5) a single gRNA targeting Cblb;(6) 2 gRNAs targeting the Zc3h12a gene and Cblb gene; (7) 2 gRNAstargeting the Map4k1 gene and Cblb gene; or (8) 2 gRNAs targeting theNr4a3 gene and Cblb gene.

Recipient CD45.2 C57BL/6J mice are injected subcutaneously with 0.5×10⁶B16-Ova tumor cells. When tumors reached a volume of approximately 100mm³, mice are randomized into groups of 10 and injected intravenouslywith edited CD45.1 TILs via tail vein. Optionally, mice can be injectedintraperitoneal with cyclophosphamide (200 mg/kg) to inducelymphodepletion prior to T cell transfer and the edited-TILs can beadministered intravenously in combination with intraperitoneal treatmentwith recombinant human IL-2 (720,000 IU/Kg) twice daily for up to amaximum of 4 days.

Body weight and tumor volume are measured at least twice per week. Tumorvolume is calculated as mean and standard error of the mean for eachtreatment group and the % TGI is calculated according to the followingformula:%TGI=(TV-target_(final))−TV-target_(initial))/(TV-Control_(final)−TV-Control_(initial)),

where TV=mean tumor volume, final=Day 17 and initial=day of edited TILtransfer.

These results are expected to show that compared to a control guide,adoptive transfer of single-edited or dual-edited mouse TILs results inan enhanced anti-tumor response in the B16Ova subcutaneous mouse modelcompared to treatment with control-edited cells.

Example 21: Validation of Targets for Engineered T Cell Therapy

Experiments will be performed to validate the effects of editingBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and/or NR4A3 on theanti-tumor efficacy of CAR T cells and T cells engineered to express anartificial TCR. The engineered T cells described in Table 17 are editedas described in Example 1 to reduce expression of BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,GNAS, ZC3H12A, MAP4K1, and/or NR4A3. These edited T cells are thenevaluated in subcutaneous murine xenograft models using the indicatedcell type. For example, T cells engineered with a CD19-specific CAR orartificial TCR can be evaluated as described above in Example 12 in aRaji cell model or any of the other cell lines shown in Table 16, Tcells engineered with a MART1-specific CAR or artificial TCR can beevaluated in a SKMEL5, WM2664, or IGR1 cell model, etc.

TABLE 16 Engineered Receptor Specificity and Target Cell Lines ReceptorSpecificity Target Cell Line CD19 Raji, Daudi, Jeko, NALM-6, NALM-16,RAMOS, JeKo1 BCMA Multiple Myeloma cell lines NCI-H929, U266-B1, andRPMI-8226 NYESO A375 MART1 SKMEL5, WM2664, IGR1 HER2+ BT474

Briefly, 6-8 week old female NSG mice from Jackson labs are injectedsubcutaneously with 3×10⁶ target cells. When tumors reached a volume ofapproximately 200 mm³, mice are randomized into groups of 5 and injectedintravenously with the edited engineered T cells via tail vein. Prior toinjection the adoptively transferred cells are edited with either acontrol guide or a guide editing for BCL2L11, FLI1, CALM2, DHODH, UMPS,RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A,MAP4K1, and/or NR4A3. Body weight and tumor volume are measured at leasttwice per week. Tumor volume is calculated as mean and standard error ofthe mean for each treatment group. The results of these experiments areexpected to show enhanced anti-tumor efficacy of BCL2L11, FLI1, CALM2,DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3,GNAS, ZC3H12A, MAP4K1, and/or NR4A3-edited engineered T cells or ascompared to a control guide, measured by survival and or reduction intumor size.

Example 22: Validation of Target Editing on Receptor-Engineered TFunction

Experiments will be performed to validate the effects of editingBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and/or NR4A3 on engineeredT cell cytokine production. Briefly, the engineered T cells described inTable 16 above are generated from human CD8 T cells, and one or more ofBCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2, PCBP1, PBRM1,WDR6, E2F8, SERPINA3, GNAS, ZC3H12A, MAP4K1, and/or NR4A3 are edited byelectroporation using guide RNAs complexed to Cas9 in an RNP format.CAR-Ts are co-cultured with the corresponding cell line indicated inTable 18 in vitro at a 1:0, 0.3:1, 1:1, 3:1 and 10:1 ratio. After 24hours, total cell counts of engineered T cells are determined, andsupernatants saved for cytokine analyses. The results of theseexperiments are expected to show enhanced accumulation of and increasedlevels of cytokine production from edited CAR T cells compared tocontrol edited cells.

Example 23: Manufacturing of Dual-Edited Tumor Infiltrating Lymphocytes

Edited TILs are manufactured following established protocols usedpreviously in FDA-approved clinical trials for the isolation andexpansion of TIL's. Following removal of tumor tissue, the tumor is bothfragmented into 2 mm³ pieces and mechanically/enzymatically homogenizedand cultured in 6,000 IU/mL recombinant human IL-2 for up to 6 weeks oruntil the cell numbers reach or exceed 1×10⁸; this is defined as thepre-rapid expansion phase (pre-REP) of TIL manufacturing. Uponcompletion of the pre-REP stage TILs are electroporated with gRNA/Cas9RNP complexes targeting BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39,SEMA7A, CHIC2, PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, ZC3H12A,MAP4K1, and/or NR4A3 genes under cGMP conditions. Cells may be alsoelectroporated prior to or during the pre-REP process. Followingelectroporation, 50×10⁶ cells are transferred into a 1 L G-Rex™ cultureflask with a 1:100 ratio of TIL:irradiated feeder cells forapproximately 2 weeks. This portion of manufacturing is defined as therapid expansion phase (REP). After the REP phase, TIL's are harvested,washed, and suspended in a solution for immediate infusion into thepatient.

Example 24: Phase I Studies of Edited Immune Effector Cells

Phase 1, open-label, single-center studies will be performed, in whichmetastatic melanoma patients who are relapsed or refractory to anti PD-1therapy will be treated with the modified cells described herein.Patients will receive a single infusion of cells and will remain onstudy until they experience progressive disease or therapy intolerance.Radiological PD will be determined by a local radiologist beforediscontinuation of study participation.

Study Objectives: The primary objectives of the study are (1) todetermine the maximum tolerated dose (MTD), dose limiting toxicities(DLTs), and dose of cell compositions (and the associated concomitantmedications required) recommended for future studies for patients withadvanced solid tumors; and (2) to observe patients for any evidence ofanti-cancer activity of the transferred edited cells. The secondaryobjectives of the study are: (1) to determine the pharmacokinetics ofthe cellular compositions; (2) to assess of on-target activity of thecellular compositions, as determined by changes in pharmacodynamicbiomarkers in biologic samples; and (3) to assess of proliferation ofthe modified cells, as determined by engineered TIL persistence posttreatment. The exploratory objectives of the study are (1) to correlateany underlying genetic mutation(s) with clinical response.

Study End-Points: The primary endpoints of this study are: Incidence andseverity of adverse events (AEs), graded according the National CancerInstitute Common Terminology Criteria for Adverse Events (NCI CTCAE),version 4.3; Clinical laboratory abnormalities; Changes in 12-leadelectrocardiogram (ECG) parameters; Objective response rate (ORR), perRECIST v1.1; CNS response (ORR and progression free survival [PFS], perRECIST v1.1, in patients who have active brain metastases). Thesecondary endpoints of this study are: Patient-reported symptoms andhealth-related quality of life (HRQoL) scores; Time to response;Duration of response; Disease control rate (the percentage of patientswith best response of complete response [CR], PR, or SD), per RECISTv1.1; Time on treatment; Immunophenotyping; Persistence, trafficking andfunction of genetically engineered TIL; Pharmacodynamic biomarker in preand post-dose samples. The exploratory endpoints of this study are:Assessment of cancer-associated mutations and/or genetic alterationsutilizing FoundationOne® Cancer Gene Panel, or comparable alternative,in pre-dose tumor biopsy and/or peripheral blood.

Treatment Regimen: A summary of the treatment regimen is as follows:

(a) Day −7 & −6: cyclophosphamide 60 mg/kg, i.v.

(b) Days −5 to −1: fludarabine 25 mg/m², i.v.

(c) Day 1: Cell infusion

(d) Day 1-Day 15: IL-2 (125,000 IU/kg/day) up to a maximum of 14administrations

The first dose of cells administered will not exceed a total dose of1×10⁹ cells. Should the patient experience dose limiting toxicity (DLT),two additional patients will be treated at this dose level. If the firstpatient completes the DLT monitoring period (21 days) withoutexperiencing a DLT, subsequent patients will be treated at doses not toexceed 1×10¹¹ TILs.

Concomitant Treatment: Palliation and supportive care are permittedduring the study for management of symptoms and underlying medicalconditions that may develop during the study.

Efficacy Evaluation: Tumor response will be determined per RECIST v1.1by the local radiologist and/or investigator. Tumor assessment will beperformed every 6 weeks until disease progression and will continue forpatients who have discontinued due to reasons other than diseaseprogression, until disease progression, or to the start of anotheranticancer therapy. Survival will also be followed for up to 3 yearsafter the last patient enrolled into the study.

Safety Evaluation: Safety assessments will include physical andlaboratory examinations, vital signs, and ECGs. Adverse events will begraded according to the NCI CTCAE v4.03. Adverse event incidence rates,as well as the frequency of occurrence of overall toxicity, categorizedby toxicity grades (severity), will be described for each cohort of thestudy. Listings of laboratory test results will also be generated, anddescriptive statistics summarizing the changes in laboratory tests overtime will be presented.

Molecular Genetic Evaluations: The mutation status of genes implicatedin tumor biology will be determined through molecular analysis of tumortissue and plasma samples. Results of these tests will be provided tothe investigator and the sponsor immediately after analysis, per thetesting procedure. Molecular analysis methods include, but are notlimited to, direct sequencing and/or digital polymerase chain reaction(PCR).

Patient-Reported Symptoms and Quality of Life Evaluations:Patient-reported symptoms and HRQoL will be collected by administeringthe validated European Organisation for Research and Treatment of Cancer(EORTC) Quality of Life Questionnaire (QLQ)-C30 (v.3.0), which has beenstudied extensively in global clinical studies. The EORTC QLQ-C30 willbe scored for 5 functional scales (physical, role, cognitive, emotional,and social functioning); 3 symptom scales (fatigue, pain, andnausea/vomiting); and a global health status/QoL scale. Six single-itemscales also are included (dyspnea, insomnia, appetite loss,constipation, diarrhea, and financial difficulties).

Study Assessments: Assessment parameters for these studies includeradiological imaging of the tumor prior to dose administration and onDay 1 of every odd number cycle thereafter, blood sample collection forPK analysis (Day 1, 2, 3, weekly×4, monthly×6) and pharmacodynamicsanalysis, and cytokine panel analysis (Day 1, 2, 3, weekly×4,monthly×6).

Example 25: Validation of Zc3h12a as Target Conferring Anti-Tumor Memoryand Epitope Spreading

Anti-tumor memory and epitope spreading driven by Zc3h12a inhibition ordeletion in T cells was evaluated in mice using the B16-Ova subcutaneoussyngeneic tumor model. 6-8 week old female C57BL/6J mice from Jacksonlabs were injected subcutaneously with 0.5×10⁶ B16-Ova tumor cells. Whentumors reached a volume of approximately 100 mm³, mice were randomizedinto groups of 10 and injected intravenously with edited mouse OT1 CD8+T cells via tail vein. Prior to injection, these cells were edited byelectroporation with gRNA/Cas9 RNP complexes comprising (i) a controlgRNA; or (ii) a single gRNA targeting the Zc3h12a gene. Body weight andtumor volume were measured at least twice per week. Tumor volume wascalculated as mean and standard error of the mean for each treatmentgroup.

As shown in FIG. 20, 9 of the 10 mice receiving Zc3h12a-edited T cellswere evaluable and rejected the tumors completely by 55 days after thetransfer of OT1 CD8+ T cells (or 65 days after the initial B16-Ova tumorinoculation). These mice were deemed to be Complete Responders (CRs).Four of the CR mice were re-challenged with a second B16-Ovainoculation, with another four CR mice challenged with a B16F10inoculation. B16-Ova is a derivative of B16F10 engineered to contain theOva antigen against which OT1 cells are specific for. In addition, tennaïve mice were inoculated with B16-Ova, and another ten naïve mice wereinoculated with B16F10 tumor cells. Following these inoculations,B16-Ova CR mice were found to also reject both B16-Ova re-challenge andB16F10 challenge. The B16-Ova rejection demonstrates a Zc3h12a-drivenanti-tumor memory response. The B16F10 rejection demonstrates thatZc3h12a-edited T cells instruct other T cells within the host to mount aproductive anti-tumor response against non-Ova tumor antigens through aphenomenon known as “epitope spreading.” Epitope spreading, which isalso known as determinant spreading, is when T cells not involved in theinitial immune response are activated against epitopes linked in atissue-specific manner to the initial response progressively contributeto an immune response.

Example 26: Editing of MAP4K1 in Naive Human T Cells Enhances CytokineProduction

Primary human T cells were isolated from thawed donor PBMCs and culturedovernight in Immunocult+10 ng/mL IL-2. The next day, T cells wereelectroporated with an RNP for a single guide control gene or MAP4K1.After four days in culture (Immunocult+10 ng/mL IL-2) to ensure geneediting, cells were cultured for 24 hr in Immunocult+CD3/28/2activation. Supernatants were collected 24 hrs after activation andsecreted cytokine levels were measured by the mesoscale discovery (MSD)platform. As shown in FIG. 21, MAP4K1-edited T cells demonstratedincreased production of IFNγ, IL-2, and TNFα. Similar experiments can beperformed to assess cytokine production of ZC3H12a-edited andNR4A3-edited PBMCs.

Example 27: Expression of T Cell Activation Markers in Zc3h12a-EditedMurine Cd8 T Cells

Spleens from female OT1 or pMEL mice were harvested and CD8 T cells wereisolated as described in Example 1. CD8 T cells were electroporated withRNPs comprising a Cas9 protein and sgRNAs targeting either Zc3h12a or acontrol gene were according to methods described in Example 1. Acrossmultiple guides, Zc3h12a-editing efficiency was measured to be 36-69%.

Expression of Ifng, Gzma, and Icos was measured by RNA-seq. RNAextraction and sequencing (RNA-seq) from pellets of 3 million editedcells was performed by Wuxi NextCode. Gene expression levels arequantified as TPMs (Li et al., in Bioinformatics: The Impact of AccurateQuantification on Proteomic and Genetic Analysis and Research (2014))using Salmon (version 0.11.2, Patro et al., Nat. Methods (2017) andGencode mouse gene annotation (version M15). R package limma³ (version3.38.0, Ritchie et al., Nucleic Acids Res. (2015)) was used to analyzethe differentially expressed genes (DEGs). When multiple guides wereused to inhibit a target, the analysis incorporated the impact of eachguide and only genes affected by all guides were consideredsignificantly differentially expressed.

These experiments revealed that Zc3h12a-edited mouse OT-1 or PMEL cellsdemonstrated significantly increased expression of Ifng (144.5-fold inOT-1; 119.2-fold in PMEL relative to control sgRNA) and Gzma mRNA(7.2-fold in OT-1 and 3.1 fold in pMEL compared to control sgRNA). InZc3h12a-edited cells, mRNA expression of Icos, a known ZC3H12Asubstrate, was also upregulated 11-fold and 9.1 fold in OT-1 and PMELcells, respectively. Together these data demonstrate that Zc3h12a-editedmouse OT-1 or PMEL cells demonstrate increased expression of the T cellactivation markers Ifng, Gzma, and Icos as measured by mRNA expression.

Example 28: Icos Expression by Zc3h12a siRNA Modified Murine T Cells

Zc3h12a mRNA targeting siRNAs were used to demonstrate the effects ofmodifying expression of the Zc3h12a by additional mechanisms ofinhibition. Control (catalog #: K-005000-G1-02) or Zc3h12a-targeting(catalog #: E-052076-00-0005) Self-delivering Accell siRNAs wereprepared according to the manufacturer's instructions. Purified murineCD8 T-cells were activated with anti-CD3/anti-CD28 beads (Dynabeads™Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation Cat#11456D) in siRNA delivery media (Dharmacon Catalog #B-005000-500)containing 2.5% heat inactivated FBS supplemented with 10 ng/mL ofrecombinant murine IL-2 (Biolegend Catalog #575406) and Self-deliveringAccell siRNAs at a final concentration of 1 μM. After 72 hours,activation beads were removed and cells were assessed for surface ICOSexpression by flow cytometry or gene expression analysis by qRT-PCR.

For qRT-PCR analysis, RNA was purified from T-cells using a QiagenRNeasy miniprep kit (catalog #: 74104) and cDNA was prepared usingSuperScript IV VILO (catalog #: 11755-050, Life Technologies)) accordingto the manufacturer's instructions. Listed below are the taqman probes(Life Technologies) used for detection of mouse or human transcripts.GAPDH was used as the control gene and relative expression wasquantified using the delta-delta Ct method. Treatment of cells withZc3h12a-specific siRNAs resulted in a 30% reduction in Zc3h12a mRNAlevels compared to controls.

Gene Mouse Assay ID Human Assay ID GAPDH Mm99999915_g1 Hs02786624_g1 IL6Mm00446190-m1 Hs00174131_m1 ICOS Mm00497600_m1 N/A GZMA Mm013044562-m1N/A IFNG Mm01168134_m1 Hs00989291_m1

For ICOS surface expression, murine CD8 cells were stained withfluorescently labeled anti-ICOS antibodies (Biolegend cat #: 313524) ata 1:100 dilution according to the manufacturer's instructions. Therelative amount of cell surface ICOS expression was measured bycalculating the mean fluorescence intensity of the cells stained withthe fluorescent anti-ICOS antibody, using FlowJo v10.1 software(Treestar). Consistent with sgRNA-mediated inhibition of Zc3h12a, ICOSlevels were 1.8-fold higher in cells treated with Zc3h12a-targetingsiRNA.

Example 29: Expression of T Cell Activation Markers in Murine T CellsEdited with Zc3h12a Zinc Fingers

ZFN-mediated editing of the Zc3h12a gene was performed to demonstratethe effects of modifying expression of the Zc3h12a by additionalmechanisms of inhibition.

Engineered zinc finger nuclease (ZFN) domains were generated by SigmaAldrich in plasmid pairs (CSTZFN-1KT COMPOZRO Custom Zinc FingerNuclease (ZFN) R-3257609). The domains were customized to recognizepositions Chr4:125122398-125122394 and Chr4:125121087-125121084 of mouseZc3h12a gene and positions Chr6:42538446-42538447 of the control mousegene Olfr455. Plasmids were prepared using the commercial NEB MonarchMiniprep system (Cat #T1010) following manufacturer's protocol. The DNAtemplate was linearized using 10 μg total input and purified using theNEB Monarch PCR and DNA Cleanup kit (Cat #T1030). An in vitrotranscription reaction to generate 5′-capped RNA transcripts wasperformed using 6 μg of purified DNA template and the Promega T7 RiboMAXLarge Scale RNA Production System (P1300 and P1712) following themanufacture's conditions. Transcripts were purified using Qiagen RNeasyMini purification kit (Cat #74104). The integrity and concentration ofeach ZFN domain transcript were confirmed using the Agilent 4200TapeStation system. Purified transcripts were polyadenylated using theNEB E. coli Poly(A) Polymerase (M0276) using 10 units per reaction. Theaddition of polyadenylated tails were confirmed by a size shift usingthe Agilent 4200 TapeStation system. Each mature ZFN domain mRNAtranscript was combined with its corresponding pair and 10 μg of eachpair mixed with 5 million mouse CD8 T cells and electroporated accordingto the methods described above for murine T cell electroporation.

Two loci in Zc3h12a were targeted by 3 ZFN each and editing efficiencywas measured by NGS. To follow the effects of ZFN-mediated ZC3H12Ainactivation, mRNA levels of the ZC3H12A substrates 116 and Icos, aswell as the T-cell activation marker Ifng, were assessed by by qRT-PCR.All 3 ZFNs targeting locus Chr4:125121088-125121085 resulted inincreased expression of Icos (range 1.2-2-fold), 116 (range2.3-62.8-fold) and Ifng (2.9-12.8) mRNA compared to a control ZFN targetOlfr455, confirming that ZFN-mediated editing of Zc3h12a induces similareffects on T-cells as CRISPR-mediated inhibition.

Example 30: Expression of T Cell Activation Markers in Zc3h12a-EditedMurine TILs

Mouse CD8 TILs were isolated and edited with Zc3h12a-targeting gRNA/Cas9RNPs as described in Example 1. Zc3h12a-specific sgRNAs lead to 86%inhibition of Zc3h12a. FACS analysis confirmed that inhibition ofZc3h12a with sgRNA led to a 2.7-fold increase in ICOS expression on thecell surface. RNA-seq analysis confirmed a significant 2.7-fold increasein Icos mRNA (p=1.6×10⁻⁷¹) as well as significant increases in the mRNAof Ifng (9.3-fold relative to control sgRNA, p=0) and Gzma (1.2 foldrelative to control sgRNA, p=6.3×10⁻⁶), confirming Zc3h12a editing inmouse TILs activates T-cells.

Example 31: Inactivation of Zc3h12a Activity by CRISPR-Mediated Editingof Zc3h12a in Mouse Cd8 T Cells

The effect of genetic inactivation of Zc3h12a on the function andinflammatory phenotype of mouse CD8 T cells was assessed. Mouse OT-I CD8T cells were activated and infected with one of 3 different retroviralsupernatants using the methods described in Example 30. Retrovirusesencoded either 1) an sgRNA targeting an olfactory receptor gene(negative control), 2) an sgRNA targeting Zc3h12a (Zc3h12a-editedcondition), or 3) an sgRNA targeting Zc3h12a along with gene encodingthe protein product of Zc3h12a (Zc3h12a edited, WT ZC3H12A rescuecondition). Post selection transduction rates were greater than 91% inall cases, and next generation sequencing confirmed that genome editingrates were greater than 80% in all cases.

CD8 T cells were then stimulated for 6 hours stimulation with PMA andIonomycin to induce inflammatory transcript expression, and quantitativeRT-PCR was performed to assess mRNA levels. Transcript expression levelswere evaluated relative to expression of the housekeeping gene Gapdh.Editing of the Zc3h12a locus led to a dramatic increase in the levels ofIcos, Il6, Il2, Ifng, and Nfkbiz transcripts when compared to negativecontrol cells edited at the Olf421 locus (FIG. 22). Expression ofZC3H12A protein by lentiviral co-delivery of a codon optimized Zc3h12agene into endogenous Zc3h12a edited cells led to a reduction ofinflammatory mRNA levels (FIG. 22), demonstrating dependence of theobserved phenotype on the absence of functional ZC3H12A protein.

To determine if the ZC3H12A-dependent transcriptional changes observedtranslated to changes on the cell surface, cells were stained withfluorescent anti-ICOS antibodies and cell surface ICOS levels weredetermined using flow cytometry. Editing of the Zc3h12a locus led to anincrease in cell surface Icos expression that was reversed byoverexpression of retrovirally derived ZC3H12A (FIG. 23).

The effects of genetic inactivation of the Zc3h12a gene on cytokinesecretion by CD8 T cells were also measured. Cells were seeded intowells containing media containing either IL-2 alone, or IL-2 andanti-CD3/CD28 dynabeads, for 48 hours. Cell supernatants were harvestedand the levels of secreted IFNγ and IL-6 were measured using themesoscale discovery MSD platform. Upon stimulation, Zc3h12a edited CD8 Tcells produced greater quantities of IL-6 and IFNγ than Olf421-editedcells. Furthermore, Zc3h12a-edited cells also produced significantquantities of IFNγ in the absence of exogenous anti-CD3/CD28 stimulationcompared to Olf421 edited cells (FIG. 24).

Example 32: Inactivation of Zc3h12a by CRISPR-Mediated Editing of theZc3h12a Gene in Primary Human T Cells

To determine the effects of inactivation of ZC3H12A on primary human Tcells, the production of IL6 mRNA transcript and IL-6 protein bygenome-edited human T cells was evaluated. Briefly, primary human Tcells were isolated from peripheral blood mononuclear cells (PBMC) andgenome-edited using RNPs targeting either the ZC3H12A or OR1A2 loci,following the methods as outline in Example 1. Editing efficienciesranged from 30-72%, as determined by next generation sequencing.

Cells were seeded at equal concentrations and cultured in culture mediumcontaining anti-CD3/CD2/CD28 tetramer reagent (Stemcell Technologies).After 24 hours, cell supernatants was harvested and IL-6 levels weredetermined using the Mesoscale discovery V-plex human proinflammatorypanel 1. Across multiple donors, T cells edited at the ZC3H12A locussecreted more IL-6 into the culture medium than those edited at theOR1A2 locus over the 24 hour period (FIG. 25A). Significantly increasedproduction of IL-6 mRNA (12.3-fold and 5.9-fold in donors 3835 and 6915,respectively) was also observed after 24 hour anti-CD3/CD28 stimulation(FIG. 25B).

Example 33: ZC3H12A-TILING SCREEN AND VALIDATION ASSAYS

A CRISPR-Cas9 tiling screen was performed to determine candidateinhibitor target locations within a target locus spanning the ZC3H12Agene. Primary human CD8+ T cells were isolated as described in Example 1and transduced with a lentiviral library expressing sgRNAs designed totarget genomic positions across the full length of the ZC3H12A gene. Twodays after transduction with the lentiviral library, the transduced CD8+T cells were electroporated with Cas9 mRNA and cultured for anadditional 10 days. After 10 days of culture subsequent toelectroporation with Cas9 mRNA, the screen performed as described below.Additional assays were performed to validate gRNAs identified in thetiling screen. 437 distinct ZC3H12A-targeting gRNAs were assayedaccording to the following parameters.

Tiling Screen

Proliferation Read-out: Cells in the first arm were harvested on Day 10after Cas9 mRNA electroporation. DNA was extracted and ampliconsspanning the recognition sites for the various sgRNAs in the librarywere amplified by PCR and sequenced by next-generation sequencing (NGS).Enrichment or depletion of each of the sgRNAs was determined by the logratio of final counts divided by reference counts.

Additional Assays for Guide Validation

Additional assays were developed to further characterize the efficacyand on-target activity of gRNAs identified in the tiling screendescribed above. Cells were isolated and electroporated with Cas9:gRNARNPs containing ZC3H12A-specific gRNAs identified in tiling screens.Cells were cultured for approximately 10 days at which point thefollowing assays were performed.

DNA Editing Assay: A DNA editing assay was developed to compare theability of individual guides to direct editing of their respectivetarget at the DNA level. After electroporation with Cas9:gRNA RNPs,cells were cultured for approximately 10 days, at which point pelletswere harvested and DNA was extracted. Amplicons spanning the genomictarget loci for the various sgRNAs were amplified by PCR usingguide-specific primer sets and sequenced by NGS. Sequencing reads werealigned to the predicted guide cut site, and the percentage of readsdisplaying an edited DNA sequence was determined. Outcomes wereevaluated based on two criteria: 1) the overall percentage of off targetediting and 2) the identity of the off-target edited genes. Optimalguides were identified those having the lowest level percent off-targetediting and/or most benign off target edited genes profile. For example,gene editing in intragenic regions was viewed as a benign effect whileediting in known oncogenes or tumor suppressors was viewed as anundesirable off target profile

Western Blot Assay: A Western Blot assay was used to compare the abilityof individual guides to reduce protein expression of their respectivetargets. P After electroporation with Cas9:gRNA RNPs, cells werecultured for approximately 10 days, cell pellets were then harvested,and lysed in RIPA buffer with protease and phosphatase inhibitors.Extracted protein was quantified by Bradford assay, and 1 μg was loadedonto an automated Western Blotting instrument (Wes Separation Module byProtein Simple) using the machine's standard 12-230 kD Wes SeparationModule protocol. Commercially available target specific primaryantibodies were employed, followed by incubation with HRP-conjugatedsecondary antibodies. The signal detected per target guide wasnormalized to the respective signal seen in the negative control guidesample.

Tiling and Validation Results: 200 ZC3H12A-targeting gRNAs (SEQ ID NOs:1065-1264) were identified as optimal guides based on the assaysdescribed above. Similar experiments were performed for BCOR andTNFAIP3, with 57 BCOR gRNAs (SEQ ID NOs: 708-764) and 39 TNFAIP3 gRNAs(SEQ ID NOs: 348-386) identified as potential optimal guides.

TABLE 5A Human Genome Coordinates Target Coordinates Target CoordinatesTarget Coordinates IKZF1 chr7: 50387344- TNFAIP3 chr6: 137879270- HAVCR2chr5: 157106863- 50387363 137879289 157106882 IKZF1 chr7: 50400471-TNFAIP3 chr6: 137878846- HAVCR2 chr5: 157088943- 50400490 137878865157088962 IKZF1 chr7: 50327652- TNFAIP3 chr6: 137876140- HAVCR2 chr5:157106706- 50327671 137876159 157106725 IKZF1 chr7: 50400507- TNFAIP3chr6: 137878571- HAVCR2 chr5: 157106886- 50400526 137878590 157106905IKZF1 chr7: 50376576- TNFAIP3 chr6: 137878573- HAVCR2 chr5: 157106767-50376595 137878592 157106786 IKZF1 chr7: 50400314- TNFAIP3 chr6:137878653- HAVCR2 chr5: 157106825- 50400333 137878672 157106844 IKZF1chr7: 50327681- TNFAIP3 chr6: 137878827- HAVCR2 chr5: 157106718-50327700 137878846 157106737 IKZF1 chr7: 50391851- TNFAIP3 chr6:137878726- HAVCR2 chr5: 157104727- 50391870 137878745 157104746 IKZF1chr7: 50368009- TNFAIP3 chr6: 137871457- HAVCR2 chr5: 157087278-50368028 137871476 157087297 IKZF1 chr7: 50382569- TNFAIP3 chr6:137876104- LAG3 chr12: 6774679- 50382588 137876123 6774698 IKZF1 chr7:50376631- TNFAIP3 chr6: 137878762- LAG3 chr12: 6773300- 50376650137878781 6773319 IKZF1 chr7: 50400366- TNFAIP3 chr6: 137876083- LAG3chr12: 6773939- 50400385 137876102 6773958 IKZF1 chr7: 50391772- TNFAIP3chr6: 137871402- LAG3 chr12: 6775340- 50391791 137871421 6775359 IKZF1chr7: 50399915- TNFAIP3 chr6: 137871501- LAG3 chr12: 6773781- 50399934137871520 6773800 IKZF1 chr7: 50400414- TNFAIP3 chr6: 137874861- LAG3chr12: 6773221- 50400433 137874880 6773240 IKZF1 chr7: 50368040- TNFAIP3chr6: 137871362- LAG3 chr12: 6773335- 50368059 137871381 6773354 IKZF1chr7: 50382550- TNFAIP3 chr6: 137871249- LAG3 chr12: 6774608- 50382569137871268 6774627 IKZF1 chr7: 50387353- TNFAIP3 chr6: 137874972- LAG3chr12: 6775514- 50387372 137874991 6775533 NFKBIA chr14: 35404635-TNFAIP3 chr6: 137878495- LAG3 chr12: 6773804- 35404654 137878514 6773823NFKBIA chr14: 35402653- TNFAIP3 chr6: 137874842- LAG3 chr12: 6773283-35402672 137874861 6773302 NFKBIA chr14: 35402494- TNFAIP3 chr6:137876139- LAG3 chr12: 6774798- 35402513 137876158 6774817 NFKBIA chr14:35404445- TNFAIP3 chr6: 137871437- TIGIT chr3: 114307905- 35404464137871456 114307924 NFKBIA chr14: 35403152- TANK chr2: 161232836- TIGITchr3: 114295774- 35403171 161232855 114295793 NFKBIA chr14: 35403258-TANK chr2: 161179709- TIGIT chr3: 114295717- 35403277 161179728114295736 NFKBIA chr14: 35404463- TANK chr2: 161224725- TIGIT chr3:114295630- 35404482 161224744 114295649 NFKBIA chr14: 35403202- TANKchr2: 161204665- TIGIT chr3: 114295615- 35403221 161204684 114295634NFKBIA chr14: 35404411- TANK chr2: 161161386- TIGIT chr3: 114295821-35404430 161161405 114295840 NFKBIA chr14: 35402666- TANK chr2:161231124- TIGIT chr3: 114295767- 35402685 161231143 114295786 NFKBIAchr14: 35403330- TANK chr2: 161179740- TIGIT chr3: 114299648- 35403349161179759 114299667 NFKBIA chr14: 35403695- TANK chr2: 161232788- TIGITchr3: 114295577- 35403714 161232807 114295596 BCL3 chr19: 44757097- TANKchr2: 161232777- TIGIT chr3: 114295650- 44757116 161232796 114295669BCL3 chr19: 44757336- TANK chr2: 161223970- TIGIT chr3: 114294023-44757355 161223989 114294042 BCL3 chr19: 44756280- TANK chr2: 161203501-TIGIT chr3: 114299682- 44756299 161203520 114299701 BCL3 chr19:44748932- TANK chr2: 161203590- CTLA4 chr2: 203872820- 44748951161203609 203872839 BCL3 chr19: 44756229- TANK chr2: 161204749- CTLA4chr2: 203871417- 44756248 161204768 203871436 BCL3 chr19: 44751352- TANKchr2: 161179691- CTLA4 chr2: 203870885- 44751371 161179710 203870904BCL3 chr19: 44756315- TANK chr2: 161161378- CTLA4 chr2: 203867944-44756334 161161397 203867963 BCL3 chr19: 44757028- FOXP3 chrX: 49258389-CTLA4 chr2: 203871421- 44757047 49258408 203871440 BCL3 chr19: 44748876-FOXP3 chrX: 49255792- CTLA4 chr2: 203872759- 44748895 49255811 203872778BCL3 chr19: 44758720- FOXP3 chrX: 49253120- CTLA4 chr2: 203867944-44758739 49253139 203867963 BCL3 chr19: 44756334- FOXP3 chrX: 49251742-CTLA4 chr2: 203870640- 44756353 49251761 203870659 BCL3 chr19: 44751300-FOXP3 chrX: 49256916- CTLA4 chr2: 203870767- 44751319 49256935 203870786BCL3 chr19: 44758258- FOXP3 chrX: 49254054- CTLA4 chr2: 203868001-44758277 49254073 203868020 IKZF3 chr17: 39765927- FOXP3 chrX: 49258314-CTLA4 chr2: 203870606- 39765946 49258333 203870625 IKZF3 chr17:39766306- FOXP3 chrX: 49251666- CTLA4 chr2: 203872716- 39766325 49251685203872735 IKZF3 chr17: 39788315- FOXP3 chrX: 49257496- PTPN6 chr12:6955147- 39788334 49257515 6955166 IKZF3 chr17: 39832082- FOXP3 chrX:49258351- PTPN6 chr12: 6956188- 39832101 49258370 6956207 IKZF3 chr17:39766366- IKZF2 chr2: 213057056- PTPN6 chr12: 6952101- 39766385213057075 6952120 IKZF3 chr17: 39766410- IKZF2 chr2: 213056895- PTPN6chr12: 6954832- 39766429 213056914 6954851 IKZF3 chr17: 39765981- IKZF2chr2: 213007992- PTPN6 chr12: 6951504- 39766000 213008011 6951523 IKZF3chr17: 39766262- IKZF2 chr2: 213022029- PTPN6 chr12: 6951637- 39766281213022048 6951656 IKZF3 chr17: 39766122- IKZF2 chr2: 213148620- PTPN6chr12: 6952004- 39766141 213148639 6952023 IKZF3 chr17: 39777926- IKZF2chr2: 213049845- PTPN6 chr12: 6954960- 39777945 213049864 6954979 IKZF3chr17: 39777960- IKZF2 chr2: 213049749- PTPN6 chr12: 6945764- 39777979213049768 6945783 IKZF3 chr17: 39791548- IKZF2 chr2: 213013838- PTPN6chr12: 6952156- 39791567 213013857 6952175 IKZF3 chr17: 39791554- IKZF2chr2: 213147704- PTPN6 chr12: 6951688- 39791573 213147723 6951707 IKZF3chr17: 39788306- IKZF2 chr2: 213007950- PTPN6 chr12: 6952055- 39788325213007969 6952074 IKZF3 chr17: 39777690- IKZF2 chr2: 213049803- PTPN6chr12: 6952004- 39777709 213049822 6952023 SMAD2 chr18: 47869428- IKZF2chr2: 213022103- PTPN6 chr12: 6954869- 47869447 213022122 6954888 SMAD2chr18: 47896710- IKZF2 chr2: 213013910- BCOR chrX: 40074116- 47896729213013929 40074135 SMAD2 chr18: 47869333- IKZF2 chr2: 213056913- BCORchrX: 40073790- 47869352 213056932 40073809 SMAD2 chr18: 47869252- IKZF2chr2: 213147790- BCOR chrX: 40077875- 47869271 213147809 40077894 SMAD2chr18: 47869371- IKZF2 chr2: 213049707- BCOR chrX: 40052324- 47869390213049726 40052343 SMAD2 chr18: 47870547- GATA3 chr10: 8064032- BCORchrX: 40073729- 47870566 8064051 40073748 SMAD2 chr18: 47896523- GATA3chr10: 8064079- BCOR chrX: 40054273- 47896542 8064098 40054292 SMAD2chr18: 47845647- GATA3 chr10: 8073748- BCOR chrX: 40073193- 478456668073767 40073212 SMAD2 chr18: 47896640- GATA3 chr10: 8058824- BCOR chrX:40074630- 47896659 8058843 40074649 TGFBR1 chr9: 99128854- GATA3 chr10:8058443- BCOR chrX: 40062797- 99128873 8058462 40062816 TGFBR1 chr9:99137867- GATA3 chr10: 8069573- BCOR chrX: 40072605- 99137886 806959240072624 TGFBR1 chr9: 99128995- GATA3 chr10: 8069532- BCOR chrX:40073675- 99129014 8069551 40073694 TGFBR1 chr9: 99132565- GATA3 chr10:8055748- BCOR chrX: 40073080- 99132584 8055767 40073099 TGFBR1 chr9:99137897- GATA3 chr10: 8058395- BCOR chrX: 40074432- 99137916 805841440074451 TGFBR1 chr9: 99137998- GATA3 chr10: 8058737- BCOR chrX:40074150- 99138017 8058756 40074169 TGFBR1 chr9: 99137939- GATA3 chr10:8058349- BCOR chrX: 40073363- 99137958 8058368 40073382 TGFBR1 chr9:99132706- GATA3 chr10: 8058824- BCOR chrX: 40064581- 99132725 805884340064600 TGFBR1 chr9: 99128942- RC3H1 chr1: 173946812- BCOR chrX:40062765- 99128961 173946831 40062784 TGFBR1 chr9: 99129014- RC3H1 chr1:173992926- BCOR chrX: 40072562- 99129033 173992945 40072581 TGFBR2 chr3:30650327- RC3H1 chr1: 173980872- BCOR chrX: 40072987- 30650346 17398089140073006 TGFBR2 chr3: 30650394- RC3H1 chr1: 173982779- BCOR chrX:40075168- 30650413 173982798 40075187 TGFBR2 chr3: 30671914- RC3H1 chr1:173980941- BCOR chrX: 40073376- 30671933 173980960 40073395 TGFBR2 chr3:30671753- RC3H1 chr1: 173992844- BCOR chrX: 40073489- 30671772 17399286340073508 TGFBR2 chr3: 30672089- RC3H1 chr1: 173992895- BCOR chrX:40072671- 30672108 173992914 40072690 TGFBR2 chr3: 30623239- RC3H1 chr1:173992882- BCOR chrX: 40073707- 30623258 173992901 40073726 TGFBR2 chr3:30650357- RC3H1 chr1: 173961717- BCOR chrX: 40072455- 30650376 17396173640072474 TGFBR2 chr3: 30672412- RC3H1 chr1: 173984495- BCOR chrX:40073856- 30672431 173984514 40073875 TGFBR2 chr3: 30671782- RC3H1 chr1:173980811- BCOR chrX: 40073454- 30671801 173980830 40073473 TGFBR2 chr3:30644886- RC3H1 chr1: 173964926- BCOR chrX: 40073223- 30644905 17396494540073242 TGFBR2 chr3: 30671709- RC3H1 chr1: 173982894- BCOR chrX:40057164- 30671728 173982913 40057183 TGFBR2 chr3: 30671765- TRAF6chr11: 36501306- BCOR chrX: 40063694- 30671784 36501325 40063713 TGFBR2chr3: 30623229- TRAF6 chr11: 36490635- BCOR chrX: 40073114- 3062324836490654 40073133 TGFBR2 chr3: 30671933- TRAF6 chr11: 36498527- BCORchrX: 40063765- 30671952 36498546 40063784 TGFBR2 chr3: 30644834- TRAF6chr11: 36492548- BCOR chrX: 40074230- 30644853 36492567 40074249 TNIP1chr5: 151039096- TRAF6 chr11: 36501355- BCOR chrX: 40063788- 15103911536501374 40063807 TNIP1 chr5: 151039165- TRAF6 chr11: 36501423- BCORchrX: 40073550- 151039184 36501442 40073569 TNIP1 chr5: 151033531- TRAF6chr11: 36501487- BCOR chrX: 40072510- 151033550 36501506 40072529 TNIP1chr5: 151052229- TRAF6 chr11: 36490112- BCOR chrX: 40074371- 15105224836490131 40074390 TNIP1 chr5: 151056754- TRAF6 chr11: 36498546- BCORchrX: 40062953- 151056773 36498565 40062972 TNIP1 chr5: 151063682- TRAF6chr11: 36490590- BCOR chrX: 40071047- 151063701 36490609 40071066 TNIP1chr5: 151033527- TRAF6 chr11: 36501262- BCOR chrX: 40073673- 15103354636501281 40073692 TNIP1 chr5: 151056795- TRAF6 chr11: 36497165- BCORchrX: 40074756- 151056814 36497184 40074775 TNIP1 chr5: 151033778- CBLBchr3: 105853475- BCOR chrX: 40074952- 151033797 105853494 40074971 TNIP1chr5: 151045881- CBLB chr3: 105853600- BCOR chrX: 40063752- 151045900105853619 40063771 TNIP1 chr5: 151063608- CBLB chr3: 105720111- BCORchrX: 40062753- 151063627 105720130 40062772 TNIP1 chr5: 151035692- CBLBchr3: 105867412- BCOR chrX: 40073052- 151035711 105867431 40073071 TNIP1chr5: 151056834- CBLB chr3: 105867529- BCOR chrX: 40075122- 151056853105867548 40075141 TNIP1 chr5: 151064993- CBLB chr3: 105720160- BCORchrX: 40063806- 151065012 105720179 40063825 TNIP1 chr5: 151033749- CBLBchr3: 105853421- BCOR chrX: 40074193- 151033768 105853440 40074212TNFAIP3 chr6: 137878782- CBLB chr3: 105751453- BCOR chrX: 40074839-137878801 105751472 40074858 TNFAIP3 chr6: 137874872- CBLB chr3:105693541- BCOR chrX: 40074647- 137874891 105693560 40074666 TNFAIP3chr6: 137878447- CBLB chr3: 105867449- BCOR chrX: 40070980- 137878466105867468 40070999 TNFAIP3 chr6: 137878901- CBLB chr3: 105853514- BCORchrX: 40074386- 137878920 105853533 40074405 TNFAIP3 chr6: 137880092-PPP2R2D chr10: 131940160- BCOR chrX: 40072494- 137880111 13194017940072513 TNFAIP3 chr6: 137878710- PPP2R2D chr10: 131934499- BCOR chrX:40074087- 137878729 131934518 40074106 TNFAIP3 chr6: 137877173- PPP2R2Dchr10: 131947775- BCOR chrX: 40057291- 137877192 131947794 40057310TNFAIP3 chr6: 137878510- PPP2R2D chr10: 131945305- BCOR chrX: 40073603-137878529 131945324 40073622 TNFAIP3 chr6: 137879002- PPP2R2D chr10:131911562- BCOR chrX: 40074157- 137879021 131911581 40074176 TNFAIP3chr6: 137871467- PPP2R2D chr10: 131944056- BCOR chrX: 40075017-137871486 131944075 40075036 TNFAIP3 chr6: 137879001- PPP2R2D chr10:131945382- BCOR chrX: 40074903- 137879020 131945401 40074922 TNFAIP3chr6: 137875731- PPP2R2D chr10: 131947633- BCOR chrX: 40074949-137875750 131947652 40074968 TNFAIP3 chr6: 137875820- PPP2R2D chr10:131901284- BCOR chrX: 40053888- 137875839 131901303 40053907 TNFAIP3chr6: 137880133- PPP2R2D chr10: 131911594- BCOR chrX: 40074785-137880152 131911613 40074804 TNFAIP3 chr6: 137878796- NRP1 chr10:332541O3- BCOR chrX: 40077894- 137878815 33254122 40077913 TNFAIP3 chr6:137877195- NRP1 chr10: 33263822- BCOR chrX: 40076456- 137877214 3326384140076475 TNFAIP3 chr6: 137880103- NRP1 chr10: 33263660- BCOR chrX:40062904- 137880122 33263679 40062923 TNFAIP3 chr6: 137875750- NRP1chr10: 33256447- PDCD1 chr2: 241852282- 137875769 33256466 241852301TNFAIP3 chr6: 137878979- NRP1 chr10: 33263677- PDCD1 chr2: 241852278-137878998 33263696 241852297 TNFAIP3 chr6: 137880119- NRP1 chr10:33263699- PDCD1 chr2: 241852879- 137880138 33263718 241852898 TNFAIP3chr6: 137878741- NRP1 chr10: 33256400- PDCD1 chr2: 241852752- 13787876033256419 241852771 TNFAIP3 chr6: 137878795- NRP1 chr10: 33254025- PDCD1chr2: 241852618- 137878814 33254044 241852637 TNFAIP3 chr6: 137878817-NRP1 chr10: 33330718- PDCD1 chr2: 241852729- 137878836 33330737241852748 TNFAIP3 chr6: 137878974- NRP1 chr10: 33254069- PDCD1 chr2:241852687- 137878993 33254088 241852706 TNFAIP3 chr6: 137874868- NRP1chr10: 33256432- PDCD1 chr2: 241852796- 137874887 33256451 241852815TNFAIP3 chr6: 137876091- HAVCR2 chr5: 157106936- PDCD1 chr2: 241852933-137876110 157106955 241852952 TNFAIP3 chr6: 137877199- HAVCR2 chr5:157095368- PDCD1 chr2: 241852831- 137877218 157095387 241852850 HAVCR2chr5: 157106898- PDCD1 chr2: 241851189- 157106917 241851208

TABLE 5B Murine Genome Coordinates Target Coordinates Target CoordinatesTarget Coordinates Ikzf1 chr11: 11754053- Gata3 chr2: 9874375- Lag3chr6: 124908392- 11754072 9874394 124908411 Ikzf1 chr11: 11707883- Gata3chr2: 9858592- Lag3 chr6: 124909391- 11707902 9858611 124909410 Ikzf1chr11: 11754068- Gata3 chr2: 9877463- Lag3 chr6: 124909410- 117540879877482 124909429 Ikzf1 chr11: 11754134- Gata3 chr2: 9877514- Tigitchr16: 43662107- 11754153 9877533 43662126 Ikzf1 chr11: 11754153- Gata3chr2: 9858607- Tigit chr16: 43662060- 11754172 9858626 43662079 Ikzf1chr11: 11754103- Gata3 chr2: 9877338- Tigit chr16: 43661976- 117541229877357 43661995 Ikzf1 chr11: 11754015- Gata3 chr2: 9863114- Tigitchr16: 43662254- 11754034 9863133 43662273 Ikzf1 chr11: 11754119- Gata3chr2: 9858626- Tigit chr16: 43661994- 11754138 9858645 43662013 Nfkbiachr12: 55491236- Rc3h1 chr1: 160930251- Tigit chr16: 43662156- 55491255160930270 43662175 Nfkbia chr12: 55491172- Rc3h1 chr1: 160930280- Tigitchr16: 43662277- 55491191 160930299 43662296 Nfkbia chr12: 55491206-Rc3h1 chr1: 160930154- Tigit chr16: 43662012- 55491225 16093017343662031 Nfkbia chr12: 55490633- Rc3h1 chr1: 160942614- Tigit chr16:43664036- 55490652 160942633 43664055 Nfkbia chr12: 55491112- Rc3h1chr1: 160930266- Tigit chr16: 43664057- 55491131 160930285 43664076Nfkbia chr12: 55490800- Rc3h1 chr1: 160930185- Tigit chr16: 43649030-55490819 160930204 43649049 Nfkbia chr12: 55490821- Rc3h1 chr1:160938126- Tigit chr16: 43662129- 55490840 160938145 43662148 Nfkbiachr12: 55490526- Rc3h1 chr1: 160930198- Tigit chr16: 43662059- 55490545160930217 43662078 Nfkbia chr12: 55491657- Traf6 chr2: 101688485- Tigitchr16: 43662148- 55491676 101688504 43662167 Nfkbia chr12: 55491177-Traf6 chr2: 101691455- Tigit chr16: 43664021- 55491196 10169147443664040 Nfkbia chr12: 55491675- Traf6 chr2: 101688575- Ctla4 chr1:60914621- 55491694 101688594 60914640 Nfkbia chr12: 55490773- Traf6chr2: 101684742- Ctla4 chr1: 60909166- 55490792 101684761 60909185Nfkbia chr12: 55490809- Traf6 chr2: 101688539- Ctla4 chr1: 60914725-55490828 101688558 60914744 Nfkbia chr12: 55491735- Traf6 chr2:101691482- Ctla4 chr1: 60909219- 55491754 101691501 60909238 Nfkbiachr12: 55490571- Traf6 chr2: 101688558- Ctla4 chr1: 60914673- 55490590101688577 60914692 Nfkbia chr12: 55490588- Traf6 chr2: 101684510- Ctla4chr1: 60912501- 55490607 101684529 60912520 Nfkbia chr12: 55491715- Cblbchr16: 52152499- Ctla4 chr1: 60912446- 55491734 52152518 60912465 Nfkbiachr12: 55492316- Cblb chr16: 52139574- Ctla4 chr1: 60912725- 5549233552139593 60912744 Nfkbia chr12: 55491207- Cblb chr16: 52139603- Ctla4chr1: 60912516- 55491226 52139622 60912535 Bcl3 chr3: 19809245- Cblbchr16: 52112122- Ctla4 chr1: 60912664- 19809264 52112141 60912683 Bcl3chr3: 19811059- Cblb chr16: 52112134- Ctla4 chr1: 60912477- 1981107852112153 60912496 Bcl3 chr3: 19809632- Cblb chr16: 52152535- Ctla4 chr1:60912618- 19809651 52152554 60912637 Bcl3 chr3: 19809634- Cblb chr16:52142891- Ctla4 chr1: 60912682- 19809653 52142910 60912701 Bcl3 chr3:19809551- Cblb chr16: 52135797- Ctla4 chr1: 60912697- 19809570 5213581660912716 Bcl3 chr3: 19809516- Cblb chr16: 52131105- Ctla4 chr1:60912605- 19809535 52131124 60912624 Bcl3 chr3: 19812411- Cblb chr16:52112169- Ctla4 chr1: 60912433- 19812430 52112188 60912452 Bcl3 chr3:19811610- Cblb chr16: 52204542- Ctla4 chr1: 60909202- 19811629 5220456160909221 Ikzf3 chr11: 98516898- Cblb chr16: 52131058- Ctla4 chr1:60909165- 98516917 52131077 60909184 Ikzf3 chr11: 98467268- Cblb chr16:52135876- Ctla4 chr1: 60914619- 98467287 52135895 60914638 Ikzf3 chr11:98467464- Cblb chr16: 52135763- Ctla4 chr1: 60909244- 98467483 5213578260909263 Ikzf3 chr11: 98467325- Cblb chr16: 52139509- Ptpn6 chr6:124727399- 98467344 52139528 124727418 Ikzf3 chr11: 98467181- Ppp2r2dchr7: 138876553- Ptpn6 chr6: 124732470- 98467200 138876572 124732489Ikzf3 chr11: 98477038- Ppp2r2d chr7: 138882200- Ptpn6 chr6: 124732484-98477057 138882219 124732503 Ikzf3 chr11: 98466977- Ppp2r2d chr7:138876565- Ptpn6 chr6: 124727385- 98466996 138876584 124727404 Ikzf3chr11: 98467103- Ppp2r2d chr7: 138882451- Ptpn6 chr6: 124721816-98467122 138882470 124721835 Tgfbr1 chr1: 47396418- Ppp2r2d chr7:138882404- Ptpn6 chr6: 124725324- 47396437 138882423 124725343 Tgfbr1chr4: 47396363- Ppp2r2d chr7: 138869675- Ptpn6 chr6: 124732430- 47396382138869694 124732449 Tgfbr1 chr4: 47393272- Ppp2r2d chr7: 138876686-Ptpn6 chr6: 124732454- 47393291 138876705 124732473 Tgfbr1 chr4:47393468- Ppp2r2d chr7: 138874130- Ptpn6 chr6: 124732329- 47393487138874149 124732348 Tgfbr1 chr4: 47393456- Nrp1 chr8: 128363358- Ptpn6chr6: 124725334- 47393475 128363377 124725353 Tgfbr1 chr4: 47396564-Nrp1 chr8: 128363296- Ptpn6 chr6: 124732349- 47396583 128363315124732368 Tgfbr1 chr4: 47393315- Nrp1 chr8: 128359628- Ptpn6 chr6:124732309- 47393334 128359647 124732328 Tgfbr1 chr1: 47396434- Nrp1chr8: 128476138- Ptpn6 chr6: 124727402- 47396453 128476157 124727421Tgfbr1 chr4: 47393288- Nrp1 chr8: 128363272- Ptpn6 chr6: 124732435-47393307 128363291 124732454 Tgfbr1 chr4: 47396512- Nrp1 chr8:128359612- Pdcd1 chr1: 94041239- 47396531 128359631 94041258 Tgfbr1chr4: 47402873- Nrp1 chr8: 128363336- Pdcd1 chr1: 94041292- 47402892128363355 94041311 Tgfbr1 chr4: 47396539- Nrp1 chr8: 128363210- Pdcd1chr1: 94041357- 47396558 128363229 94041376 Tgfbr1 chr1: 47393266- Nrp1chr8: 128425932- Pdcd1 chr1: 94041207- 47393285 128425951 94041226Tgfbr1 chr4: 47396394- Nrp1 chr8: 128497936- Pdcd1 chr1: 94041223-47396413 128497955 94041242 Tgfbr1 chr1: 47393462- Nrp1 chr8: 128468551-Pdcd1 chr1: 94041394- 47393481 128468570 94041413 Tgfbr2 chr9:116129944- Nrp1 chr8: 128363251- Pdcd1 chr1: 94041165- 116129963128363270 94041184 Tgfbr2 chr9: 116129900- Nrp1 chr8: 128460693- Pdcd1chr1: 94041179- 116129919 128460712 94041198 Tgfbr2 chr9: 116129928-Havcr2 chr11: 46456439- Pdcd1 chr1: 94041468- 116129947 4645645894041487 Tgfbr2 chr9: 116131548- Havcr2 chr11: 46469515- Pdcd1 chr1:94041331- 116131567 46469534 94041350 Tgfbr2 chr9: 116131562- Havcr2chr11: 46466864- Pdcd1 chr1: 94041421- 116131581 46466883 94041440Tgfbr2 chr9: 116131610- Havcr2 chr11: 46479374- Pdcd1 chr1: 94041165-116131629 46479393 94041184 Tgfbr2 chr9: 116131588- Havcr2 chr11:46456495- Pdcd1 chr1: 94041421- 116131607 46456514 94041440 Tgfbr2 chr9:116131529- Havcr2 chr11: 46479356- Pdcd1 chr1: 94041331- 11613154846479375 94041350 Tgfbr2 chr9: 116110272- Havcr2 chr11: 46455033- Pdcd1chr1: 94041468- 116110291 46455052 94041487 Tgfbr2 chr9: 116109969-Havcr2 chr11: 46469534- Pdcd1 chr1: 94041239- 116109988 4646955394041258 Tgfbr2 chr9: 116129901- Havcr2 chr11: 46456242- Pdcd1 chr1:94041292- 116129920 46456261 94041311 Tgfbr2 chr9: 116129988- Havcr2chr11: 46479302- Pdcd1 chr1: 94041357- 116130007 46479321 94041376Tgfbr2 chr9: 116110004- Havcr2 chr11: 46456496- Pdcd1 chr1: 94041207-116110023 46456515 94041226 Tnip1 chr11: 54939673- Havcr2 chr11:46456355- Pdcd1 chr1: 94041223- 54939692 46456374 94041242 Tnip1 chr11:54930778- Havcr2 chr11: 46469521- Pdcd1 chr1: 94041394- 5493079746469540 94041413 Tnip1 chr11: 54934036- Havcr2 chr11: 46459111- Pdcd1chr1: 94041179- 54934055 46459130 94041198 Tnip1 chr11: 54934071- Havcr2chr11: 46456301- Pdcd1 chr1: 94041412- 54934090 46456320 94041431 Tnip1chr11: 54930799- Lag3 chr6: 124908571- Pdcd1 chr1: 94041268- 54930818124908590 94041287 Tnip1 chr11: 54930820- Lag3 chr6: 124909259- Pdcd1chr1: 94041309- 54930839 124909278 94041328 Tnip1 chr11: 54933977- Lag3chr6: 124909424- Pdcd1 chr1: 94041469- 54933996 124909443 94041488 Tnip1chr11: 54929117- Lag3 chr6: 124908491- Pdcd1 chr1: 94041189- 54929136124908510 94041208 Tnfaip3 chr10: 19011464- Lag3 chr6: 124909299- Pdcd1chr1: 94041331- 19011483 124909318 94041350 Tnfaip3 chr10: 19008246-Lag3 chr6: 124909474- Pdcd1 chr1: 94041239- 19008265 124909493 94041258Tnfaip3 chr10: 19008332- Lag3 chr6: 124909286- Pdcd1 chr1: 94041292-19008351 124909305 94041311 Tnfaip3 chr10: 19006919- Lag3 chr6:124908450- 19006938 124908469 Tnfaip3 chr10: 19008294- Lag3 chr6:124908529- 19008313 124908548 Tnfaip3 chr10: 19008234- Lag3 chr6:124909272- 19008253 124909291 Tnfaip3 chr10: 19002796- Lag3 chr6:124909399- 19002815 124909418 Tnfaip3 chr10: 19006981- Lag3 chr6:124909228- 19007000 124909247

TABLE 6A Human Genome Coordinates Target Coordinates Target CoordinatesTarget Coordinates BCL2L11 chr2: 111123809- PBRM1 chr3: 52554752- CALM2chr2: 47167608- 111123828 52554771 47167627 BCL2L11 chr2: 111142346-PBRM1 chr3: 52603635- CALM2 chr2: 47162389- 111142365 52603654 47162408BCL2L11 chr2: 111150125- PBRM1 chr3: 52634703- CALM2 chr2: 47162623-111150144 52634722 47162642 BCL2L11 chr2: 111164161- PBRM1 chr3:52662232- CALM2 chr2: 47161766- 111164180 52662251 47161785 BCL2L11chr2: 111123880- PBRM1 chr3: 52609796- CALM2 chr2: 47161806- 11112389952609815 47161825 BCL2L11 chr2: 111142303- PBRM1 chr3: 52554720- CALM2chr2: 47162544- 111142322 52554739 47162563 BCL2L11 chr2: 111128637-PBRM1 chr3: 52668623- CALM2 chr2: 47167482- 111128656 52668642 47167501BCL2L11 chr2: 111124067- PBRM1 chr3: 52679663- CALM2 chr2: 47162606-111124086 52679682 47162625 BCL2L11 chr2: 111150032- PBRM1 chr3:52617272- CALM2 chr2: 47162351- 111150051 52617291 47162370 BCL2L11chr2: 111153772- PBRM1 chr3: 52678502- CALM2 chr2: 47162279- 11115379152678521 47162298 BCL2L11 chr2: 111124106- PBRM1 chr3: 52558272- CALM2chr2: 47172416- 111124125 52558291 47172435 BCL2L11 chr2: 111123866-PBRM1 chr3: 52668512- SERPINA3 chr14: 94614673- 111123885 5266853194614692 BCL2L11 chr2: 111130128- PBRM1 chr3: 52643284- SERPINA3 chr14:94619278- 111130147 52643303 94619297 BCL2L11 chr2: 111123761- PBRM1chr3: 52558266- SERPINA3 chr14: 94614582- 111123780 52558285 94614601BCL2L11 chr2: 111150081- PBRM1 chr3: 52634800- SERPINA3 chr14: 94619423-111150100 52634819 94619442 BCL2L11 chr2: 111123790- PBRM1 chr3:52603596- SERPINA3 chr14: 94614528- 111123809 52603615 94614547 BCL2L11chr2: 111153779- PBRM1 chr3: 52643330- SERPINA3 chr14: 94614599-111153798 52643349 94614618 BCL2L11 chr2: 111124008- PBRM1 chr3:52651751- SERPINA3 chr14: 94614744- 111124027 52651770 94614763 BCL2L11chr2: 111123848- WDR6 chr3: 49008972- SERPINA3 chr14: 94614944-111123867 49008991 94614963 BCL2L11 chr2: 111123849- WDR6 chr3:49011963- SERPINA3 chr14: 94614885- 111123868 49011982 94614904 CHIC2chr1: 54064267- WDR6 chr3: 49011741- SERPINA3 chr14: 94614692- 5406428649011760 94614711 CHIC2 chr1: 54049066- WDR6 chr3: 49014895- SEMA7Achr15: 74417586- 54049085 49014914 74417605 CHIC2 chr1: 54048982- WDR6chr3: 49012228- SEMA7A chr15: 74416690- 54049001 49012247 74416709 CHIC2chr1: 54064276- WDR6 chr3: 49007462- SEMA7A chr15: 74417405- 5406429549007481 74417424 CHIC2 chr4: 54014101- WDR6 chr3: 49012620- SEMA7Achr15: 74416640- 54014120 49012639 74416659 CHIC2 chr4: 54013870- WDR6chr3: 49012948- SEMA7A chr1 5: 74415947- 54013889 49012967 74415966CHIC2 chr1: 54049029- RBM39 chr20: 35729298- SEMA7A chr1 5: 74411646-54049048 35729317 74411665 CHIC2 chr1: 54049258- RBM39 chr20: 35738973-SEMA7A chr15: 74417429- 54049277 35738992 74417448 CHIC2 chr1: 54064203-RBM39 chr20: 35725067- SEMA7A chr15: 74414850- 54064222 3572508674414869 CHIC2 chr1: 54064222- RBM39 chr20: 35714187- SEMA7A chr15:74417393- 54064241 35714206 74417412 CHIC2 chr4: 54014065- RBM39 chr20:35716784- DHODH chr16: 72014466- 54014084 35716803 72014485 CHIC2 chr1:54064183- RBM39 chr20: 35739528- DHODH chr16: 72008782- 5406420235739547 72008801 FLI1 chr11: 128772938- RBM39 chr20: 35734223- DHODHchr16: 72012120- 128772957 35734242 72012139 FLI1 chr11: 128810556-RBM39 chr20: 35735042- DHODH chr16: 72012061- 128810575 3573506172012080 FLI1 chr11: 128768268- RBM39 chr20: 35724711- DHODH chr16:72022430- 128768287 35724730 72022449 FLI1 chr11: 128772807- RBM39chr20: 35729482- DHODH chr16: 72014503- 128772826 35729501 72014522 FLI1chr11: 128807189- RBM39 chr20: 35731997- DHODH chr16: 72014529-128807208 35732016 72014548 FLI1 chr11: 128768230- RBM39 chr20:35731969- DHODH chr16: 72012094- 128768249 35731988 72012113 FLI1 chr11:128807207- RBM39 chr20: 35740826- DHODH chr16: 72012147- 12880722635740845 72012166 FLI1 chr11: 128810519- RBM39 chr20: 35716771- DHODHchr16: 72017036- 128810538 35716790 72017055 FLI1 chr11: 128810490-RBM39 chr20: 35707976- DHODH chr16: 72008781- 128810509 3570799572008800 FLI1 chr11: 128810665- RBM39 chr20: 35734220- DHODH chr16:72012216- 128810684 35734239 72012235 FLI1 chr11: 128772978- RBM39chr20: 35707942- DHODH chr16: 72014491- 128772997 35707961 72014510 FLI1chr11: 128772894- RBM39 chr20: 35729478- DHODH chr16: 72008781-128772913 35729497 72008800 PCBP1 chr2: 70087872- RBM39 chr20: 35740555-DHODH chr16: 72014548- 70087891 35740574 72014567 PCBP1 chr2: 70087909-RBM39 chr20: 35736543- UMPS chr3: 124738139- 70087928 35736562 124738158PCBP1 chr2: 70087790- RBM39 chr20: 35739531- UMPS chr3: 124730574-70087809 35739550 124730593 PCBP1 chr2: 70087821- RBM39 chr20: 35732003-UMPS chr3: 124737663- 70087840 35732022 124737682 PCBP1 chr2: 70087998-RBM39 chr20: 35714241- UMPS chr3: 124737918- 70088017 35714260 124737937PCBP1 chr2: 70088588- RBM39 chr20: 35736551- UMPS chr3: 124735177-70088607 35736570 124735196 PCBP1 chr2: 70088106- E2F8 chr11: 19234923-GNAS chr20: 58895661- 70088125 19234942 58895680 PCBP1 chr2: 70087940-E2F8 chr11: 19234390- GNAS chr20: 58903685- 70087959 19234409 58903704PCBP1 chr2: 70088307- E2F8 chr11: 19237345- GNAS chr20: 58905460-70088326 19237364 58905479 PCBP1 chr2: 70088200- E2F8 chr11: 19235005-GNAS chr20: 58840352- 70088219 19235024 58840371 PCBP1 chr2: 70088063-E2F8 chr11: 19225425- GNAS chr20: 58840096- 70088082 19225444 58840115PCBP1 chr2: 70087845- E2F8 chr11: 19237329- GNAS chr20: 58840253-70087864 19237348 58840272 E2F8 chr11: 19234967- GNAS chr20: 58891819-19234986 58891838 E2F8 chr11: 19234422- GNAS chr20: 58891756- 1923444158891775 E2F8 chr11: 19237906- GNAS chr20: 58891768- 19237925 58891787E2F8 chr11: 19237980- GNAS chr20: 58840195- 19237999 58840214 E2F8chr11: 19232290- GNAS chr20: 58891728- 19232309 58891747 E2F8 chr11:19229509- GNAS chr20: 58840198- 19229528 58840217

TABLE 6B Murine Genome Coordinates Target Coordinates Target CoordinatesTarget Coordinates Bcl2l11 chr2: 128128713- Fli1 chr9: 32461444- Wdr6chr9: 108578530- 128128732 32461463 108578549 BCl2l11 chr2: 128147115-Fi11 chr9: 32461386- Wdr6 chr9: 108576565- 128147134 32461405 108576584Bcl2l11 chr2: 128128731- Fli1 chr9: 32461401- Wdr6 chr9: 108578514-128128750 32461420 108578533 Bcl2l11 chr2: 128147173- Fli1 chr9:32465687- Wdr6 chr9: 108578497- 128147192 32465706 108578516 Bcl2l11chr2: 128128648- Fli1 chr9: 32461420- Wdr6 chr9: 108576511- 12812866732461439 108576530 Bcl2l11 chr2: 128128660- Fli1 chr9: 32424186- Dhodhchr8: 109596082- 128128679 32424205 109596101 Bcl2l11 chr2: 128147091-Fli1 chr9: 32461239- Dhodh chr8: 109601459- 128147110 32461258 109601478Bcl2l11 chr2: 128128682- Fli1 chr9: 32424232- Dhodh chr8: 109603453-128128701 32424251 109603472 Bcl2l11 chr2: 128128640- Pcbp1 chr6:86525508- Dhodh chr8: 109603306- 128128659 86525527 109603325 Bcl2l11chr2: 128147141- Pcbp1 chr6: 86524927- Dhodh chr8: 109603364- 12814716086524946 109603383 Bcl2l11 chr2: 128158269- Pcbp1 chr6: 86525842- Dhodhchr8: 109603351- 128158288 86525861 109603370 Bcl2l11 chr2: 128158233-Pcbp1 chr6: 86525525- Dhodh chr8: 109596173- 128158252 86525544109596192 Bcl2l11 chr2: 128147129- Pcbp1 chr6: 86525608- Dhodh chr8:109601503- 128147148 86525627 109601522 Bcl2l11 chr2: 128128753- Pcbp1chr6: 86525731- Gnas chr2: 174334196- 128128772 86525750 174334215Bcl2l11 chr2: 128158301- Pcbp1 chr6: 86525676- Gnas chr2: 174345476-128158320 86525695 174345495 Bcl2l11 chr2: 128147086- Pcbp1 chr6:86525148- Gnas chr2: 174346023- 128147105 86525167 174346042 Bcl2l11chr2: 128128730- Pbrm1 14: 31040494- Gnas chr2: 174341872- 12812874931040513 174341891 Bcl2l11 chr2: 128128992- Pbrm1 14: 31038941- Gnaschr2: 174345749- 128129011 31038960 174345768 Chic2 chr5: 75027179-Pbrm1 14: 31061547- Gnas chr2: 174345419- 75027198 31061566 174345438Chic2 chr5: 75044295- Pbrm1 14: 31036055- Gnas chr2: 174334251- 7504431431036074 174334270 Chic2 chr5: 75044192- Pbrm1 14: 31067548- Gnas chr2:174345768- 75044211 31067567 174345787 Chic2 chr5: 75011480- Pbrm1 14:31027510- 75011499 31027529 Chic2 chr5: 75044214- Pbrm1 14: 31067943-75044233 31067962 Chic2 chr5: 75011437- Pbrm1 14: 31030854- 7501145631030873 Chic2 chr5: 75027108- 75027127 Chic2 chr5: 75044244- 75044263

TABLE 6C Human Genome Coordinates Target Coordinates Target CoordinatesTarget Coordinates ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480274_37480293 37482857_37482876 37483381_37483400 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482967_37482986 37475578_3747559737482899_37482918 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482922_37482941 37480329_37480348 37480373_37480392 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37480273_37480292 37480288_3748030737481847_37481866 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482886_37482905 37481600_37481619 37483330_37483349 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483185_37483204 37483212_3748323137483065_37483084 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475817_37475836 37483337_37483356 37482499_37482518 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483033_37483052 37475542_3747556137483105_37483124 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480408_37480427 37483197_37483216 37475631_37475650 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483026_37483045 37482730_3748274937483530_37483549 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483463_37483482 37475599_37475618 37483407_37483426 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37480362_37480381 37483262_3748328137483308_37483327 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482962_37482981 37482790_37482809 37482853_37482872 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475775_37475794 37482719_3748273837482934_37482953 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475509_37475528 37482860_37482879 37475591_37475610 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475722_37475741 37483443_3748346237475826_37475845 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475818_37475837 37483558_37483577 37475865_37475884 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482966_37482985 37481599_3748161837481784_37481803 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480388_37480407 37475845_37475864 37480322_37480341 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483142_37483161 37475730_3747574937475664_37475683 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482448_37482467 37482524_37482543 37475757_37475776 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483049_37483068 37482849_3748286837483385_37483404 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482905_37482924 37475529_37475548 37482933_37482952 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482733_37482752 37475664_3747568337475866_37475885 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480423_37480442 37482972_37482991 37475843_37475862 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482456_37482475 37483321_3748334037475797_37475816 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483551_37483570 37482984_37483003 37475642_37475661 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37481767_37481786 37475807_3747582637483270_37483289 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475715_37475734 37483213_37483232 37483024_37483043 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483377_37483396 37482427_3748244637483201_37483220 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475593_37475612 37483104_37483123 37482447_37482466 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475875_37475894 37482879_3748289837483253_37483272 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475534_37475553 37483409_37483428 37483429_37483448 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482764_37482783 37482752_3748277137483195_37483214 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475869_37475888 37480391_37480410 37481648_37481667 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483437_37483456 37475694_3747571337483424_37483443 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475598_37475617 37482458_37482477 37475580_37475599 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482438_37482457 37475774_3747579337482980_37482999 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483257_37483276 37475574_37475593 37480408_37480427 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483263_37483282 37475803_3747582237483405_37483424 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482545_37482564 37481605_37481624 37475740_37475759 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483015_37483034 37482437_3748245637480387_37480406 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481595_37481614 37482825_37482844 37483507_37483526 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482923_37482942 37483595_3748361437483110_37483129 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483143_37483162 37483510_37483529 37483325_37483344 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482348_37482367 37483283_3748330237481692_37481711 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483018_37483037 37482446_37482465 37475826_37475845 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482612_37482631 37475700_3747571937483098_37483117 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475613_37475632 37475721_37475740 37481758_37481777 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475563_37475582 37475628_3747564737480320_37480339 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475535_37475554 37482848_37482867 37483380_37483399 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482843_37482862 37483134_3748315337483011_37483030 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480424_37480443 37475543_37475562 37483509_37483528 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482606_37482625 37482799_3748281837483509_37483528 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483098_37483117 37483296_37483315 37482768_37482787 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483508_37483527 37483332_3748335137475804_37475823 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483559_37483578 37483600_37483619 37475808_37475827 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483256_37483275 37482410_3748242937475859_37475878 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475936_37475955 37481718_37481737 37482973_37482992 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475607_37475626 37483395_3748341437475634_37475653 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475809_37475828 37482428_37482447 37475854_37475873 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483186_37483205 37475562_3747558137480334_37480353 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481747_37481766 37483500_37483519 37480414_37480433 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482734_37482753 37475827_3747584637480316_37480335 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483278_37483297 37483586_37483605 37482971_37482990 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482332_37482351 37483089_3748310837482781_37482800 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483109_37483128 37483419_37483438 37483173_37483192 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475633_37475652 37480285_3748030437482391_37482410 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482591_37482610 37483256_37483275 37482392_37482411 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483271_37483290 37483420_3748343937482936_37482955 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483603_37483622 37475691_37475710 37483408_37483427 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482504_37482523 37483419_3748343837481779_37481798 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483252_37483271 37475918_37475937 37483206_37483225 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483119_37483138 37475589_3747560837482561_37482580 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482343_37482362 37482362_37482381 37481745_37481764 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483144_37483163 37482566_3748258537475802_37475821 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483213_37483232 37482963_37482982 37483494_37483513 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482981_37483000 37483420_3748343937483371_37483390 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482789_37482808 37483139_37483158 37482552_37482571 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483159_37483178 37483619_3748363837475491_37475510 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482349_37482368 37481764_37481783 37482479_37482498 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483602_37483621 37475650_3747566937483140_37483159 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481596_37481615 37483405_37483424 37483313_37483332 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482537_37482556 37483037_3748305637483458_37483477 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482370_37482389 37483211_37483230 37483320_37483339 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475546_37475565 37475537_3747555637483204_37483223 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482598_37482617 37475756_37475775 37475792_37475811 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483146_37483165 37482403_3748242237483475_37483494 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475812_37475831 37482455_37482474 37475577_37475596 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483400_37483419 37480311_3748033037475787_37475806 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475703_37475722 37482586_37482605 37483574_37483593 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483418_37483437 37483099_3748311837480284_37480303 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480284_37480303 37483342_37483361 37482369_37482388 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482800_37482819 37481823_3748184237483384_37483403 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475721_37475740 37482777_37482796 37483425_37483444 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482715_37482734 37482412_3748243137482582_37482601 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37480281_37480300 37483604_37483623 37483153_37483172 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482491_37482510 37483438_3748345737482935_37482954 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483497_37483516 37482445_37482464 37483378_37483397 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475899_37475918 37483331_3748335037482952_37482971 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475889_37475908 37483111_37483130 37483399_37483418 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482375_37482394 37482847_3748286637483309_37483328 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475741_37475760 37483249_37483268 37483200_37483219 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482900_37482919 37481754_3748177337481641_37481660 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482442_37482461 37475684_37475703 37481656_37481675 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37481644_37481663 37482519_3748253837483036_37483055 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482464_37482483 37482475_37482494 37483474_37483493 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482994_37483013 37482613_3748263237483004_37483023 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483437_37483456 37482939_37482958 37481846_37481865 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482736_37482755 37475541_3747556037483205_37483224 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482538_37482557 37481763_37481782 37483406_37483425 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483515_37483534 37483231_3748325037480336_37480355 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475874_37475893 37482953_37482972 37481716_37481735 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483145_37483164 37482407_3748242637480335_37480354 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482587_37482606 37475808_37475827 37481659_37481678 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37475482_37475501 37481620_3748163937475809_37475828 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475844_37475863 37475592_37475611 37482565_37482584 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37480415_37480434 37483156_3748317537482491_37482510 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481709_37481728 37480329_37480348 37483379_37483398 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483366_37483385 37475573_3747559237481654_37481673 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475627_37475646 37483198_37483217 37482567_37482586 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482447_37482466 37483557_3748357637481614_37481633 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481758_37481777 37482892_37482911 37482562_37482581 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483560_37483579 37483334_3748335337475868_37475887 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475869_37475888 37481708_37481727 37482557_37482576 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37481655_37481674 37483063_3748308237483511_37483530 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37481645_37481664 37482998_37483017 37475615_37475634 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483016_37483035 37482942_3748296137483333_37483352 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475838_37475857 37475508_37475527 37482840_37482859 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37482850_37482869 37482371_3748239037483545_37483564 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475510_37475529 37483119_37483138 37482830_37482849 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483510_37483529 37482798_3748281737482444_37482463 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483064_37483083 37475859_37475878 37482571_37482590 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483149_37483168 37483401_3748342037482553_37482572 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37483449_37483468 37482851_37482870 37483543_37483562 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37483264_37483283 37475524_3747554337483542_37483561 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37475508_37475527 37475601_37475620 37482575_37482594 ZC3H12A Chr1:ZC3H12A Chr1: ZC3H12A Chr1: 37480415_37480434 37475815_3747583437475855_37475874 ZC3H12A Chr1: ZC3H12A Chr1: ZC3H12A Chr1:37482918_37482937 37482801_37482820 37482572_37482591 ZC3H12A Chr1:ZC3H12A Chr1: Zc3h12a chr1: 37482474_37482493 37475544_37475563125122335- 125122354 ZC3H12A Chr1: ZC3H12A Chr1: Zc3h12a chr1:37483232_37483251 37483010_37483029 125121083- 125121102 ZC3H12A Chr1:ZC3H12A Chr1: Zc3h12a chr1: 37475732_37475751 37483077_37483096125120961- 125120980 ZC3H12A Chr1: ZC3H12A Chr1: Zc3h12a chr1:37481602_37481621 37482404_37482423 125122390- 125122409 ZC3H12A Chr1:ZC3H12A Chr1: Zc3h12a chr1: 37480289_37480308 37475692_37475711125120373- 125120392 ZC3H12A Chr1: ZC3H12A Chr1: Zc3h12a chr1:37483165_37483184 37483596_37483615 125122250- 125122269 ZC3H12A Chr1:ZC3H12A Chr1: Zc3h12a chr1: 37483248_37483267 37483372_37483391125122375- 125122394 ZC3H12A Chr1: ZC3H12A Chr1: Zc3h12a chr1:37483078_37483097 37481596_37481615 125120975- 125120994 ZC3H12A Chr1:ZC3H12A Chr1: 37483017_37483036 37480370_37480389 ZC3H12A Chr1: ZC3H12AChr1: 37483174_37483193 37480377_37480396

TABLE 6D Murine Genome Coordinates Target Coordinates Zc3h12a chr1:125122335-125122354 Zc3h12a chr1: 125121083-125121102 Zc3h12a chr1:125120961-125120980 Zc3h12a chr1: 125122390-125122409 Zc3h12a chr1:125120373-125120392 Zc3h12a chr1: 125122250-125122269 Zc3h12a chr1:125122375-125122394 Zc3h12a chr1: 125120975-125120994

TABLE 6E Human Genome Coordinates Target Coordinates MAP4K1 chr19:38617794-38617813 MAP4K1 chr19: 38612617-38612636 MAP4K1 chr19:38599957-38599976 MAP4K1 chr19: 38617616-38617635 MAP4K1 chr19:38607904-38607923 MAP4K1 chr19: 38613865-38613884 MAP4K1 chr19:38607916-38607935 MAP4K1 chr19: 38617828-38617847 MAP4K1 chr19:38616225-38616244 MAP4K1 chr19: 38610007-38610026 MAP4K1 chr19:38599964-38599983 MAP4K1 chr19: 38612614-38612633 MAP4K1 chr19:38617365-38617384

TABLE 6F Murine Genome Coordinates Target Coordinates Map4k1 chr7:28983466-28983485 Map4k1 chr7: 28983244-28983263 Map4k1 chr7:28983478-28983497 Map4k1 chr7: 28983003-28983022 Map4k1 chr7:28983420-28983439 Map4k1 chr7: 28983436-28983455 Map4k1 chr7:28983405-28983424 Map4k1 chr7: 28983214-28983233 Map4k1 chr7:28984113-28984132 Map4k1 chr7: 28984134-28984153 Map4k1 chr7:28983019-28983038 Map4k1 chr7: 28986802-28986821 Map4k1 chr7:28983446-28983465 Map4k1 chr7: 28983220-28983239 Map4k1 chr7:28983466-28983485 Map4k1 chr7: 28983478-28983497

TABLE 6G Human Genome Coordinates Target Coordinates NR4A3 chr9:99826780-99826799 NR4A3 chr9: 99828951-99828970 NR4A3 chr9:99828105-99828124 NR4A3 chr9: 99826768-99826787 NR4A3 chr9:99828319-99828338 NR4A3 chr9: 99832766-99832785 NR4A3 chr9:99828263-99828282 NR4A3 chr9: 99828909-99828928 NR4A3 chr9:99828489-99828508 NR4A3 chr9: 99828437-99828456 NR4A3 chr9:99828669-99828688 NR4A3 chr9: 99832703-99832722

TABLE 6H Murine Genome Coordinates Target Coordinates Nr4a3 chr4:48051580-48051599 Nr4a3 chr4: 48052171-48052190 Nr4a3 chr4:48055944-48055963 Nr4a3 chr4: 48056028-48056047 Nr4a3 chr4:48051515-48051534 Nr4a3 chr4: 48052131-48052150 Nr4a3 chr4:48051303-48051322 Nr4a3 chr4: 48052115-48052134 Nr4a3 chr4:48051287-48051306 Nr4a3 chr4: 48051580-48051599 Nr4a3 chr4:48052171-48052190 Nr4a3 chr4: 48051348-48051367 Nr4a3 chr4:48051255-48051274 Nr4a3 chr4: 48051366-48051385 Nr4a3 chr4:48056024-48056043 Nr4a3 chr4: 48055983-48056002

The invention claimed is:
 1. A primary human T cell comprising (a) amodified endogenous ZC3H12A gene and (b) an engineered antigen receptorthat specifically binds to a tumor antigen, wherein ZC3H12A proteinexpression and/or function is reduced in the primary human T cell,relative to ZC3H12A protein expression and/or function in a primaryhuman T cell expressing the endogenous ZC3H12A gene.
 2. The primaryhuman T cell of claim 1, further comprising a modified endogenous targetgene selected from the group consisting of IKZF1, IKZF3, GATA3, BCL3,TNIP1, TNFAIP3, NFKBIA, SMAD2, TGFBR1, TGFBR2, TANK, FOXP3, RC3H1,TRAF6, IKZF2, CBLB, PPP2R2D, NRP1, HAVCR2, LAG3, TIGIT, CTLA4, PTPN6,PDCD1, BCOR, BCL2L11, FLI1, CALM2, DHODH, UMPS, RBM39, SEMA7A, CHIC2,PCBP1, PBRM1, WDR6, E2F8, SERPINA3, GNAS, MAP4K1 and NR4A3.
 3. Theprimary human T cell of claim 1, wherein the modified endogenous ZC3H12Agene comprises an insertion, deletion, and/or mutation.
 4. The primaryhuman T cell of claim 1, further comprising a gene-regulating system. 5.The primary human T cell of claim 1, wherein the primary human T cell isa tumor infiltrating lymphocyte.
 6. The primary human T cell of claim 1,further comprising an exogenous transgene expressing an immuneactivating molecule selected from the group consisting of a cytokine, achemokine, a co-stimulatory molecule, an activating peptide, and anantibody or an antigen-binding fragment thereof.
 7. A pharmaceuticalcomposition comprising the primary human T cell of claim
 1. 8. Theprimary human T cell of claim 1, wherein the engineered antigen receptoris a chimeric antigen receptor (CAR) or an engineered T cell receptor(TCR).
 9. The primary human T cell of claim 4, wherein the generegulating system comprises a Cas endonuclease and a guide RNA (gRNA).10. The primary human T cell of claim 9, wherein the Cas endonucleaseis: (a) a wild-type Cas protein comprising two enzymatically activedomains; (b) a Cas nickase mutant comprising one enzymatically activedomain; (c) a deactivated Cas protein (dCas) associated with aheterologous protein; or (d) a Cas ortholog.
 11. The primary human Tcell of claim 10, wherein the Cas endonuclease is a Cas9 protein. 12.The primary human T cell of claim 9, wherein the gRNA comprises atargeting domain sequence that hybridizes to a target sequence in theendogenous ZC3H12A gene.
 13. The primary human T cell of claim 12,wherein the target sequence is selected from the group consisting of SEQID NOs: 1065-1264.
 14. The primary human T cell of claim 12, wherein thegRNA targeting domain sequence is encoded by a sequence selected fromthe group consisting of SEQ ID NOs: 1065-1264.
 15. The primary human Tcell of claim 4, wherein the gene regulating system comprises (a) a zincfinger nuclease that comprises a zinc finger binding domain or (b) atranscription-activator-like effector nuclease (TALEN) that comprises atranscription-activator-like (TAL) effector domain.
 16. The primaryhuman T cell of claim 15, wherein the zinc finger binding domain or TALeffector domain binds to a target sequence in the endogenous ZC3H12Agene.
 17. The primary human T cell of claim 16, wherein the zinc fingerbinding domain or TAL effector domain binds to a target sequenceselected from the group consisting of SEQ ID NOs: 1065-1264.
 18. Theprimary human T cell of claim 4, wherein the gene regulating systemcomprises an RNA interference molecule.
 19. The primary human T cell ofclaim 18, wherein the RNA interference molecule is an siRNA, an shRNA, amicroRNA (miR), an antagomiR, or an antisense RNA.
 20. The primary humanT cell of claim 19, wherein the RNA interference molecule is an siRNA orshRNA that binds to a sequence encoded by a sequence selected from thegroup consisting of SEQ ID NOs: 1065-1264.
 21. The primary human T cellof claim 1, wherein the primary human T cell is selected from CD4+ Tcells, CD8+ T cells, Th1 cells, Th2 cells, and Th17 cells.